Patent Application
20230357757
The present invention relates to nucleic acid encoded chemical libraries, particularly self-purified nucleic acid encoded chemical libraries, and methods for production and application thereof.
DNA encoded chemical libraries (DEL) are powerful tools for drug discovery. The first methods proposed for the production of DNA-encoded chemical libraries employed alternating stepwise synthesis of a polymer (e.g. a peptide) and an oligonucleotide sequence (serving as a coding sequence) on a common linker (e.g. a bead) in split and pool cycles (Brenner, S. and Lerner, R. A. PNAS USA 89 (1992), 5381-5383; U.S. Pat. No. 5,573,905; WO93/20242). After affinity capture on a target protein, the population of identifier oligonucleotides of the selected library members would be amplified by PCR. The structures of the chemical entities would be decoded by sequencing the PCR products. It was postulated that encoding procedures could be implemented by a variety of methods, including chemical synthesis, DNA polymerization or ligation of DNA fragments (Brenner, S. and Lerner, R. A. PNAS USA 89 (1992), 5381-5383; U.S. Pat. No. 5,573,905; WO93/20242). Various methods of generating DNA-encoded chemical libraries have subsequently been described in the art (see for example Mannocci, L. et al. PNAS USA 105(46):17670-17675; Brenner, S. and Lerner, R. A. supra; Nielsen, J., et al., J. Am. Chem. Soc. 115 (1993); Needels et al., M. C., PNAS USA 90 (1993), 10700-10704; Gartner, Z. J., et al., Science 305 (2004), 1601-1605; Melkko, S., et al., Nat. Biotechnol. 22 568-574 (2004); Sprinz, K. I., et al., Bioorg. Med. Chem. Lett. 15 (2005), pp. 3908-3911; Leimbacher et al Chemistry. 2012 Jun. 18; 18(25):7729-37; Clark et al Nat Chem Biol. 2009 September; 5(9):647-54; WO2009/077173; WO2003/076943; EP3284851; EP3184674).
The established use of DEL technology enables the screening of a large number of compounds (typically in the order of 1 to 100 million) which are individually encoded by a specific nucleic acid tag and affinity-based screening of the entire DEL for a protein of interest can be performed in a single experiment. DEL technology is now widely used in the pharmaceutical industry.
To date, the variable yields of the individual synthesis steps in the construction of DELs have restricted the number of consecutive synthesis steps and the nature of building blocks which can be incorporated. Methods that allow the construction of DELs of increased size and/or purity would be useful.
The present inventors have developed a method of producing nucleic acid encoded libraries which self-purifies intact or complete library members from intermediates through selective cleavage from a solid support. This may, for example, facilitate the production of large and/or pure nucleic acid encoded libraries and/or nucleic acid encoded libraries in which the individual members have a complex structure and/or a large number of building blocks. Libraries produced by these methods may for example display improved screening performance and/or contain members capable of binding to large surfaces on target proteins.
A first aspect of the invention provides a method for producing a nucleic acid encoded compound which includes the steps of;
A second aspect of the invention provides a method for producing a nucleic acid encoded chemical library comprising, for each library member, the steps of;
In some embodiments of the first and second aspects, the cleaving group may be attached to the chemical portion (
In other embodiments of the first and second aspects, the cleaving group may be attached to the coding nucleic acid portion (
In other embodiments of the first and second aspects, the cleaving group may be attached to the scaffold (
In some embodiments of the first and second aspects, the scaffold of the nascent compound or member may be additionally connected to the solid support by a second linker, such that the scaffold is connected to the solid support by a first linker and a second linker. The method may comprise;
The first and second linkers may be cleaved sequentially in any order or simultaneously.
The compound or member is released from the solid support if it comprises (i) both a complete chemical portion or a complete segment of the chemical portion and a complete nucleic acid portion, or a complete segment of the nucleic acid portion (ii) both a complete chemical portion or a complete segment of the chemical portion and a complete scaffold, or (iii) both a complete coding nucleic acid portion, or a complete segment of the coding nucleic acid portion and a complete scaffold.
In some embodiments, the cleaving group or first cleaving group may be covalently attached to the chemical portion or the scaffold. Preferably, the cleaving group is covalently attached to the terminal chemical building block of the chemical portion.
In other embodiments, the cleaving group may be non-covalently attached to the chemical portion or scaffold. For example, by attaching an anchor oligonucleotide to the chemical portion or the scaffold, preferably to the terminal chemical building block of the chemical portion, and hybridizing said anchor oligonucleotide with an auxiliary oligonucleotide which is linked to a cleaving group. For example, a method of producing a nucleic acid encoded compound or chemical library may comprise the steps of;
In some embodiments, the cleaving group may be covalently attached to the nucleic acid portion. Preferably, the cleaving group is covalently attached to the end of the nucleic acid portion.
In other embodiments, the cleaving group may be non-covalently attached to the nucleic acid portion, for example, by hybridizing said coding nucleic acid portion with an auxiliary oligonucleotide which is linked to a cleaving group. Preferably, the auxiliary oligonucleotide is hybridized to the end of the coding nucleic acid portion. For example, a method of producing a nucleic acid encoded compound or chemical library may comprise the steps of;
The linker may be transformed or activated after attachment to the chemical portion, scaffold, or the coding nucleic acid portion and before reaction with the cleaving group.
In some embodiments, the cleaving group may not require further transformation after attachment to the chemical portion, scaffold, or the coding nucleic acid portion and before reaction with the linker. In other embodiments, the cleaving group may be activated after attachment to the chemical portion, scaffold, or the coding nucleic acid portion and before reaction with the linker.
In some preferred embodiments, a capping step may be performed after each chemical building block addition, and optionally each coding oligonucleotide addition. This prevents the cleaving group from attaching to incomplete nucleic acid or peptide nucleic acid encoded compounds.
In the aspects described herein, in addition to cleaving the linker and releasing the compound or member, the cleaving group may form a covalent bond that links the end of chemical portion to the scaffold to generate a macrocycle. For example, the reaction of the linker and the cleaving group may generate a cyclisation element or cleavage moiety that covalently links the chain of chemical building blocks to the scaffold, such that the chemical entity displayed by the member is macrocyclic.
A third aspect of the invention provides a method for producing a nucleic acid encoded compound which includes the steps of;
First and second linkers according to the third aspect may be orthogonally cleavable.
In methods of the third aspect, compounds with complete chemical portions are covalently connected to the solid support by the second linker. Compounds with incomplete chemical portions are not covalently connected to the solid support by the second linker. Compounds with incomplete chemical portions are thus selectively released from the solid support by cleavage of the first linker. Compounds with complete chemical portions remain attached to the solid support by the second linker. Cleavage of the second linker selectively releases these compounds from the solid support.
Preferably, a capping step is performed after the addition of each chemical building block. Compounds with incomplete chemical portions may remain capped to prevent connection to the second linker.
In all of the first to third aspects described herein, the chemical building blocks may be added sequentially to the nascent member or compound to form a linear chemical portion (i.e. a chain of chemical building blocks) with an end attached to the nascent member and a free end. The coding oligonucleotides encoding each chemical building block may be added sequentially to the nascent member to form a linear coding nucleic acid portion. Methods of the invention may comprise covalently attaching a chemical building block to the nascent compound or member and covalently attaching a coding oligonucleotide encoding the chemical building block to the nascent member. This may be repeated one or more times to produce the chemical portion and the coding nucleic acid portion. Following the attachment of a chemical building block, any unreacted species which lack the attached chemical building block may be capped before addition of the next chemical building block.
A fourth aspect provides a nucleic acid encoded library produced by a method of the first to the third aspects.
These and other aspects and embodiments of the invention are described in more detail below.
In some aspects, nucleic acid encoded libraries may be produced as described herein by a method that involves preparing for each library member a solid support compound comprising a scaffold attached to a solid support via a linker, a chemical portion attached to the scaffold, a nucleic portion attached to the scaffold, as well as a cleaving group attached to the chemical portion, scaffold, or nucleic acid portion. The method further comprises reacting the cleaving group with the linker to release the compound from the solid support to form the library member.
Cleavage of the linker by the cleaving group may generate a macrocycle. The macrocycle may comprise the chemical portion and the scaffold. The end of the chemical portion may be covalently connected to the scaffold in the macrocycle by a cyclisation element generated by the reaction of the cleaving group with the activated linker.
Members with a complete chemical portion, scaffold, or coding nucleic acid portion cleaving group (i.e. species in which all of the intended chemical building blocks, coding oligonucleotides or other elements are present) are selectively released from the solid support through cleavage of the linker by the cleaving group. For example, the complete chemical portion may be a chain of linked chemical building blocks (i.e. species in which all of the intended chemical building blocks are present in the chemical portion). The cleaving group does not attach to compounds or members with an incomplete or partial chemical portion, scaffold, or coding nucleic acid portion cleaving group (e.g. unreacted or partially reacted intermediates which are capped), so these compounds or members are not released from the solid support. For example, members with an incomplete or partial chemical portion that does not contain all of the intended chemical building blocks are not released from the solid support. This self-purifies complete compounds or library members and may avoid the need for further purification steps, for example using chromatographic techniques such as HPLC. Self-purification as described herein allows the production of highly pure members and facilitates the production of complex library members with many synthetic steps. The methods described herein may be rapid compared to existing DEL production techniques. They may be amenable to automation and allow the production of high quality DELs that are highly diverse.
The process of separating complete members or compounds from incompletely synthesized compounds which are not cleaved from the solid support may be referred to herein as self-purification. The compound or member which is released from the solid support after cleavage of the linker or linkers and contains the scaffold, the chemical portion, and nucleic acid portion, and may contain parts of linker and parts of cleaving group reacted together in the cleavage reaction, may be referred to as a self-purified compound or member.
In other aspects, nucleic acid encoded libraries may be produced as described herein by a method that involves preparing for each library member a solid support compound comprising a scaffold attached to a solid support via a first linker, a chemical portion attached to the scaffold, a nucleic acid portion attached to the scaffold and a second linker connecting the chemical portion to the solid support. The method further comprises cleaving the first linker, optional washing, and then cleaving the second linker to release the compound from the solid support to form the library member.
The second linker does not attach to compounds or members with an incomplete or partial chemical portion, (e.g. unreacted or partially reacted intermediates which are capped). These compounds or members are attached to the solid support only by the first linker and are released from the solid support by cleavage of the first linker. For example, members with an incomplete or partial chemical portion that does not contain all of the intended chemical building blocks are released from the solid support by cleavage of the first linker and may be removed. Members with a complete chemical portion (i.e. species in which all of the intended chemical building blocks are present) are attached to the solid support by both the first and second linkers. For example, the complete chemical portion may be a chain of linked chemical building blocks (i.e. species in which all of the intended chemical building blocks are present in the chemical portion). These members are selectively released from the solid support through cleavage of the second linker. This self-purifies complete compounds or library members and may avoid the need for further purification steps, for example using chromatographic techniques such as HPLC. Self-purification as described herein allows the production of highly pure members and facilitates the production of complex library members with many synthetic steps. The methods described herein may be rapid compared to existing DEL production techniques. They may be amenable to automation and allow the production of high quality DELs that are highly diverse.
The process of separating complete compounds from incompletely synthesized compounds which are released from the solid support by cleavage of the first linker may be referred to herein as self-purification. The compound or member which is released from the solid support after cleavage of the second linker and contains the scaffold, the complete chemical portion, and nucleic acid portion, and may contain parts of the linker retained following in the cleavage reaction, may be referred to as a self-purified compound or member.
The first and second linker may be orthogonally cleavable. For example, the reaction conditions required to cleave the first linker may be different to the reaction conditions required to cleave the second linker. The first and second linker are therefore independently cleavable by altering the reaction conditions. The first linker 1 must be stable during the synthesis of the solid support compound. The second linker must be stable to the cleavage conditions of the first linker (i.e. the second linker must not be cleaved under conditions that will cause cleavage of the first linker. The cleavage conditions of the first and second linkers must not cause degradation or destruction of the nucleic acid portion.
In some embodiments, the first and/or the second linker may be activated before cleavage.
Suitable first and/or second linkers may include base-cleavable linkers, such as ester linkers, photocleavable, amino (methyl) aniline (MeDbz), amino aniline (Dbz), masked thioester, sulfonamide, oxidatively cleavable, reductively cleavable and enzymatically cleavable linkers.
Suitable first and/or second linkers may include linkers which may be cleaved by nucleophiles. Examples of linkers which may be cleaved by nucleophiles may include thioesters, sulfonamides, benzimidazolones (for example, MeNbz), and benzotriazoles (for example, Dbz linker activated by isopentyl nitrite).
Suitable base-cleavable linkers, such as ester linkers, may be cleaved at high pH. Examples of base-based linkers may include esters, benzyl esters, and 4-(Hydroxymethyl)benzoic acid (HMBA) (Usanov, D. L. et al Nat. Chem. 10, 704-714 (2018); Soural, M. et al Linkers for Solid-Phase Peptide Synthesis. in Amino Acids, Peptides and Proteins in Organic Chemistry vol. 3 273-312 (Wiley-VCH, 2011))
Photocleavable linkers may be cleaved by photoirradiation. Examples of photocleavable linkers may include ortho-nitrobenzyloxy and ortho-nitrobenzylamino, ortho-nitrovetaryl, phenacyl, pivaloyl, benzoin linkers, and other photolabile linkers (Mikkelsen, R. J. T. et al Photolabile Linkers for Solid-Phase Synthesis. (2018) doi:10.1021/acscombsci.8b00028.)
Oxidatively cleavable linkers include geminal diols, such as linkers based on L-tartrate, seramox, and isoseramox linkers (Usanov, D. L. et al Nat. Chem. 10, 704-714 (2018); Pomplun et al Angew. Chemie—Int. Ed. 59, 11566-11572 (2020) These may be cleaved, for example, by sodium periodate.
Other suitable first and/or second linkers are available in the art and include sulfonamide linkers (Mende, F et al. J. Am. Chem. Soc. 132, 11110-11118 (2010)) and cleavable linkers (Scott, P. J. H. Linker Strategies in Solid-Phase Organic Synthesis (2009); Hermanson, G. T., Bioconjugate Techniques: Third Edition (2013); Leriche, G., Chisholm, L. & Wagner, A., Cleavable linkers in chemical biology (2012)). In some embodiments, the first and second linker may be incorporated in a single chemical entity. For example, a single first and second linker may be based on iminodiacetic acid. The first linker may be cleaved in a deprotection-mediated cyclization to yield a diketopiperazine second linker, which is then cleaved at high pH. (Pá Tek, M. & Lebl, M. Safety-Catch and Multiply Cleavable Linkers in Solid-Phase Synthesis. Biopoly vol. 47 (1998); Kočiš, P., Krchňa{acute over (k)}, V. & Lebl, Tetrahedron Lett. 34, 7251-7252 (1993)).
Other suitable first and/or second linkers may comprise two or more binding groups before attachment to the solid support and to the scaffold and/or to the chemical portion and/or to the nucleic acid portion. For example, a linker may bind to the solid support through a first binding group and may bind to the scaffold and/or to the chemical portion and/or to the nucleic acid portion through a second binding group. The binding groups of linkers may be functional groups. Suitable first and/or second linkers may be homo- or heterobifunctional, referring to their binding groups. Suitable heterobifunctional linkers may include 3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-4-(methylamino)benzoic acid (Fmoc-MeDbz-OH), wherein the carboxylic acid group may for example bind to the solid support and the amine group, after Fmoc deprotection, may for example bind to the scaffold. Other suitable heterobifunctional linkers may include 4-amino-3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]benzoic acid (Fmoc-Dbz-OH) and 4-(hydroxymethyl)benzoic acid (HMBA).
A DNA encoded chemical library (DEL) is a collection of chemically diverse library members that each comprise (i) a chemical portion formed from a set of chemically linked chemical building blocks and (ii) a nucleic acid that encodes the set of chemical building blocks that form the chemical portion. The number of different members in a library represents the complexity of a library and is defined by the number of building blocks that form each chemical portion, and by number of different variants of each building block.
A chemical portion is a chemical entity or molecular structure that is displayed by a library member and comprises one, two or more chemical building blocks. The chemical portion is covalently linked to the scaffold and is created by the consecutive covalent addition of the one or more chemical building blocks to form a linear chain or backbone having an end attached to the scaffold and a free end. A cleaving group may be attached to the chemical portion. The different chemical portions displayed by members of a DEL library are formed from different combinations of chemical building blocks. The chemical portions displayed by a DEL may be linear, macrocyclic, bicyclic, multicyclic or branched compounds of different sizes (e.g. Lipinski-like small compounds and larger compounds). In some embodiments, a chemical portion may be any small molecule (i.e. a molecule that has a molecular weight below about 1,000 Daltons). In other embodiments, a chemical portion may be any medium-sized molecule (i.e. a molecule that has a molecular weight below about 5,000 Daltons). Small molecules may be organic or inorganic, isolated (e.g., from compound libraries or natural sources), or obtained by derivatization of known compounds. A chemical portion may be designed or built to have one or more desired characteristics, e.g., capacity to bind a biological target, solubility, availability of hydrogen bond donors and acceptors, rotational degrees of freedom of the bonds, positive charge, negative charge, In vivo stability, cell permeability, and/or oral availability.
Chemical portions displayed in a nucleic acid encoded chemical library may be attached to a single strand of nucleic acid (“single pharmacophore libraries”) or two different strands of nucleic acid hybridised together, one or more building blocks being attached to each strand (“dual pharmacophore libraries”).
The coding nucleic acid portion is a nucleic acid tag or peptide nucleic acid tag which identifies the chemical building blocks in the chemical portion. The coding nucleic acid portion may be a linear nucleic acid molecule which comprises an end connected to the compound or member and a free end. Preferably, the coding nucleic acid portion is attached to the scaffold. In order to record a previously or subsequently introduced chemical building block, the coding nucleic acid portion may be elongated by covalent addition of a coding oligonucleotide to the free end. The coding nucleic acid portion can be used for the identification of the self-purified compound after amplification and nucleic acid sequencing. In some embodiments, a cleaving group may be attached to the nucleic acid portion, for example at the free end.
In the methods described herein, members of a nucleic acid encoded chemical library may be constructed on a solid support by the addition of chemical building blocks and coding oligonucleotides to nascent members.
When attached to a solid support, a nascent member may be referred to as a solid support compound. A solid support compound is a compound that comprises a solid support that is connected via the linker to the scaffold, chemical portion, nucleic acid portion, cleaving group and other elements of the nascent member. Examples of solid support compounds according to some embodiments, are shown in
Examples of self-purification reactions (i.e. the reaction of the cleaving group with the linker) are exemplified in
The reaction of the cleaving group with the linker may result in the release of a compound or member from the solid support that comprises a cleavage moiety (also referred to as a cyclisation element) formed by the reaction of the linker and the cleaving group (i.e. a cleaving group/linker product, see
In some embodiments, the cleavage moiety may be connected to the chemical portion where the cleaving group was connected to the chemical portion, and may be connected to the scaffold where the linker was connected to the scaffold. The reaction of the cleaving group with the linker may result in a cyclisation reaction, which yields a cyclic compound attached to the coding nucleic acid portion (
In other embodiments, the cleavage moiety may be connected to the chemical portion where the cleaving group was connected to the chemical portion. The reaction of the cleaving group with the linker may not yield cyclisation during the self-purification (see
Examples of solid support compounds according to other embodiments are shown in
As described above, the first and second linkers may be orthogonally cleavable.
Examples of self-purification reactions (i.e. the reaction of the cleaving group with the linker) are exemplified in
The cleavage of the first linker may result in the release from the solid support of compounds or members that have an incomplete or partial chemical portion. These compounds or members may be removed for example by washing. The cleavage of the second linker may result in the release from the solid support of compounds or members that have a complete chemical portion. The released compounds or members may comprise a first and/or a second cleavage moiety (also referred to as a cyclisation element) formed by the cleavage of the first and/or a second linker, respectively. All or part of the first and/or second linker (i.e. the cleaved linker) may remain attached to the solid support.
In some preferred embodiments, the nucleic acid encoded chemical library may be synthesized by consecutive split-and-pool steps, each step comprising the incorporation of a chemical building block to the chemical portion preceded, followed by or simultaneous with the incorporation of a coding oligonucleotide.
Individual nucleic acid encoded compounds or library members may be synthesised by a method which includes first preparing a solid support compound.
In some embodiments, the solid support compound is prepared by attaching a scaffold to a solid support through a linker, attaching a coding nucleic acid portion to the scaffold, attaching a chemical portion to the scaffold, and attaching a cleaving group to the chemical portion, scaffold, or nucleic acid portion. Following the preparation of the solid support compound, the cleaving group is reacted with the linker such that the linker is cleaved and the nucleic acid encoded compound or library member released from the solid support. For example, a nucleic acid encoded compound or library member may be produced as described herein by a method comprising the steps of;
In other embodiments, the solid support compound is prepared by attaching a scaffold to a solid support through a first linker, attaching a coding nucleic acid portion to the scaffold, attaching a chemical portion to the scaffold, and attaching a second linker to the chemical portion to connect the chemical portion to the solid support. Following the preparation of the solid support compound, the first linker is cleaved, and then the second linker is cleaved and the nucleic acid encoded compound or library member released from the solid support. For example, a nucleic acid encoded compound or library member may be produced as described herein by a method comprising the steps of;
The first chemical building block may be attached to the scaffold before, after or simultaneous with the attachment of the first coding oligonucleotide to the scaffold.
The solid support compound may be prepared, for example, by first attaching the linker to the solid support, and subsequently attaching the scaffold to the linker on the solid support; or by first attaching the scaffold to the linker to form a linker-scaffold conjugate, and subsequently attaching the linker-scaffold conjugate to the solid support.
The coding nucleic acid portion or a fragment of the coding nucleic acid portion may be attached to the scaffold before, after or simultaneous with the attachment of the scaffold to the solid support.
The chemical portion or a fragment of the chemical portion, such as a chemical building block, may be attached to the scaffold before, after or simultaneous with the attachment of the scaffold to the solid support.
A scaffold is a chemical moiety to which the chemical building blocks that form the chemical portion are attached. In some embodiments, preferably, the same chemical moiety forms the scaffold for all of the members of the library. The scaffold may be an at least trifunctional chemical moiety that connects the solid support, the nucleic acid portion, and the chemical portion.
The scaffold may comprise a capture group. The capture group is a reactive chemical group that is capable of reacting with a chemical building block to form a covalent bond linking the chemical building block to the scaffold. This allows the chemical building blocks that form the chemical portion to be attached to the scaffold.
The cleaving group is the chemical reagent or enzyme which can cleave the linker, with or without prior cleaving group activation. In order to provide self-purified compound or member, the cleaving group may in some embodiments, be either covalently or non-covalently linked to the chemical portion, the nucleic acid portion, or the scaffold. The cleaving group may need to be activated after incorporation. The compound or member may be purified by selective release from solid support through the cleavage of the linker by the cleaving group.
In some embodiments, the cleaving group may be attached to the scaffold. Compounds or library members with a complete scaffold and cleaving group may be selectively released from solid support through the cleavage of the linker by the cleaving group.
In other embodiments, the cleaving group may be attached to the chemical portion. Compounds or library members with a complete scaffold, chemical portion, and cleaving group may be selectively released from solid support through the cleavage of the linker by the cleaving group.
In other embodiments, the cleaving group may be attached to the nucleic acid portion, preferably the end of the nucleic acid portion. Compounds or library members with a complete scaffold, nucleic acid portion and cleaving group may be selectively released from solid support through the cleavage of the linker by the cleaving group.
In other embodiments, the scaffold may be connected to the solid support by two different linkers (a first and a second linker), and two different cleaving groups may be present in the solid support compound (a first cleaving group that cleaves the first linker and a second cleaving group that cleaves the second linker). One of the cleaving groups may be attached to the chemical portion or scaffold, and the other cleaving group may be attached to the nucleic acid portion. For example, the method may further comprise;
The selective release from the solid support may be initiated by the presence of both a complete chemical portion and a complete scaffold, both a complete coding nucleic acid portion and a complete scaffold, or more preferably both a complete chemical portion and a complete nucleic acid portion.
An example of the selective release of a compound or member prepared using two different linkers and two different cleaving groups is shown in
In some embodiments, the compound or member may be released from the solid support only when it contains both a complete chain of chemical building blocks of the chemical portion and a complete nucleic acid molecule comprising coding oligonucleotides for all of the chemical building blocks in the chemical portion.
In some embodiments, the cleaving group may be covalently attached to the chemical portion, or scaffold. Preferably, the cleaving group may be covalently attached to the terminal chemical building block in the chemical portion (i.e. at the free end of the chain of chemical building blocks that form the chemical portion). This allows self-purification of members or compounds comprising the whole of the chemical portion.
In other embodiments, the cleaving group may be non-covalently attached to the chemical portion or to the scaffold by hybridization of two oligonucleotides. An anchor oligonucleotide may be covalently attached to the chemical portion or scaffold, preferably the terminal chemical building block of the chemical portion. An auxiliary oligonucleotide which is covalently attached to a cleaving group may then be hybridized to the anchor oligonucleotide. This non-covalently attaches the cleaving group to the chemical portion or scaffold. The cleaving group may then react with the linker to cleave the linker and release the member or compound from solid support. For example, the cleaving group may be attached to the chemical portion or scaffold by a method comprising;
In some embodiments, the anchor oligonucleotide, which is attached to the chemical portion or scaffold, may be synthesised sequentially on the chemical portion or scaffold, respectively. The anchor oligonucleotide may also be a nucleic acid analogue, such as a peptide nucleic acid. The peptide nucleic acid may, for example, be synthesised sequentially on the scaffold or chemical portion.
Preferably, the anchor oligonucleotide may be covalently attached to the terminal chemical building block of the chemical portion.
The auxiliary oligonucleotide covalently attached to the cleaving group may be a single stranded polynucleotide that is capable of specific hybridisation to the anchor oligonucleotide. Preferably, the auxiliary oligonucleotide covalently attached to the cleaving group comprises 10 or more base pairs.
In some embodiments, the cleaving group may be covalently attached to the nucleic acid portion, for example to the free end of the nucleic acid portion.
In some preferred embodiments, the cleaving group may be linked to the coding nucleic acid portion by hybridization of an auxiliary oligonucleotide covalently attached to a cleaving group to the coding nucleic acid portion in the solid support compound. For example, the cleaving group may be attached to the coding nucleic acid portion by a method comprising;
Preferably, the auxiliary oligonucleotide is hybridized to the end portion of the coding nucleic acid (i.e. the region, segment or portion of the nucleic acid which is furthest away or distal from the scaffold). This allows self-purification of members or compounds comprising the whole of the nucleic acid portion.
The auxiliary oligonucleotide may be a single stranded polynucleotide that is capable of specific hybridisation to the coding nucleic acid portion. Preferably, the auxiliary oligonucleotide covalently attached to the cleaving group comprises 10 or more base pairs. Preferably, the auxiliary oligonucleotide covalently attached to the cleaving group hybridises to the end region of the coding nucleic acid portion, for example within 10 bases, 20 bases or 30 bases of the free end of the coding nucleic acid portion.
The coding, attachment, anchor and auxiliary oligonucleotides described herein and the coding nucleic acid portion may be, independently, a natural nucleic acid, such as DNA or RNA, or a nucleic acid analogue, such as a peptide nucleic acid (PNA), a phosphorodiamidate morpholino oligomer (PMO), a phosphorothioate oligomer (PTO), locked nucleic acid (LNA), a glycol nucleic acid (GNA) or a threose nucleic acid (TNA). The auxiliary oligonucleotide may be attached to the cleaving group by any convenient chemistry.
In other embodiments, the scaffold may be connected to the solid support by a first linker and the chemical portion may be connected to the solid support by a second linker. The first and second linker are orthogonally cleavable i.e. the first linker is cleaved under first reaction conditions and the second linker is cleaved under second reaction conditions. For example, the method may further comprise;
Suitable first and second linkers are described elsewhere herein.
Reactive groups, such as capture groups, binding groups and cleaving groups, may be protected during one or more steps in which the reactive group is not required to react. A reactive group may be conveniently protected by being covalently linked to a protecting group. The reactive group may be deprotected by removing the protecting group before a step in which the reaction of the reactive group is required.
A protecting group is a chemical group that reversibly protects a capture group, binding group, cleaving group or other reactive group described herein against undesirable reactions during one or more steps in which reaction of the capture group, binding group, cleaving group or other reactive group is not required. Commonly used protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis,” 4th Edition (John Wiley & Sons, New York, 2007). Examples of suitable protecting groups include ester groups (e.g., (methoxyethyl)ester, isovaleryl ester, and -levulinyl ester), trityl groups (e.g., dimethoxytrityl and monomethoxytrityl), xanthenyl groups (e.g., 9-20 phenylxanthen-9-yl and 9-(p-methoxyphenyl)xanthen-9-yl), acyl groups (e.g., phenoxyacetyl and acetyl), silyl groups (e.g., t-butyldimethylsilyl), 2-nitrobenzyl, allyloxycarbonyl-aminomethyl, Allocam oNv, tert-butyl group, tert-butylsulfenyl (StBu) group, sulfonate group 9-fluorenylmethyl group, 9-fluorenylmethoxycarbonyl group or an intramolecular disulfide.
For example, the capture group of the scaffold may be protected e.g. by covalent linkage to a protecting group. The capture group may be deprotected before reaction with the first chemical building block, for example by removing the protecting group.
Protecting groups may be added and removed by any convenient method. Suitable techniques are well established in the art. In some embodiments, the protecting groups may be added and removed by means of a nucleic acid compatible chemical reaction, or more generally, a reaction compatible with the encoding system. In other embodiments, protecting groups may be added or removed by enzymatic transformation. For example, an enzyme-catalysed reaction may be used to protect or deprotect binding groups in the nascent member.
Preferably, the chemical portion is produced by sequentially adding chemical building blocks to the nascent member to produce a linear sequence of chemical building blocks (i.e. a chain) that is attached at a proximal end to the scaffold. The chemical building blocks in the chain may form the chemical portion that is displayed by the member to which it is attached. For example, a first chemical building block may be covalently attached to the capture group of the scaffold. A second chemical building block may be attached to the first chemical building block to form a linear sequence or chain consisting of two chemical building blocks. The chain of chemical building blocks may have a proximal end that is attached to the scaffold and a distal end that is free. The first chemical building block may be at the proximal end position and the second chemical building block may be at the distal end position (i.e. the second chemical building block is the end or terminal chemical building block in the chain). The chain may be extended by sequential attachment of further chemical building blocks to the distal end of the chain e.g. by reaction with the chemical building block at the end position. Following completion of the chain of chemical building blocks, the cleaving group may be attached to the distal end of the complete chain, for example by reaction with the chemical building block at the distal end (the terminal chemical building block).
A chemical building block is a chemical group that forms a structural unit of a chemical portion displayed by a library member. A chemical building block may be any chemical group that comprises one, two, or more binding groups. If a chemical building block is incorporated in a chain of chemical building blocks, this chemical building block may be any chemical group that comprises two or more binding groups.
Preferably, chemical building blocks may comprise two or more binding groups that allow for covalent linkage to the scaffold or to other chemical building blocks. The two or more binding groups may display different or orthogonal reactivity. For example, a chemical building block may comprise a proximal and a distal binding group (e.g. a bifunctional building block). For example, a chemical building block may be covalently attached to the nascent member through the proximal binding group. The distal binding group of the chemical building block may be protected and/or used to attach further chemical building blocks to the nascent member. Each binding group may be a reactive functional group capable of reacting with a binding group from another chemical building block. The proximal and distal binding groups on two different building blocks; or the binding group on a building block and the capture group of the scaffold should be complementary, i.e., capable of reacting together to form a covalent bond. Any reaction compatible with the encoding system, the solid support, and linker integrity may be employed. In some embodiments, any suitable DNA compatible chemistry may be employed, for example amidation, Sonogashira coupling, Suzuki coupling, or copper (I)-catalyzed azide alkyne cycloaddition (CuAAC) or other click reactions. For example, one of the first and second binding groups may be carboxyl group and the other may be an amine group.
Suitable binding groups include carboxylic acids, alkynes, aryl halides, alkyl halides, aldehydes, ketones, nitriles, sulfonyl halides, thiols, alcohols, acetylenes, primary amines, secondary amines, azides, amidines, diamines, epoxides, isocyanates, sulfonamides and boronic acids. Suitable chemical building blocks comprising two or more binding groups include unnatural amino acids, D-amino acids, N-alkylated amino acids, and acid alkynes.
In some embodiments, chemical building blocks may comprise additional binding groups that allow cross-linking between different chemical building blocks within a chain of chemical building blocks, for example to produce macrocyclic, bicyclic or multicyclic chemical entities. For example, a trifunctional building block may be an amino acid with a side-chain with a functionality such as an alkyne, azide, amine, carboxylic acid, thiol, alcohol, or alkyl halide. In some embodiments, chemical building blocks may be crosslinked using a CuAAC reaction or other click reaction, for example between a chemical building block comprising an alkyne binding group and a chemical building block comprising an azide binding group.
In some preferred embodiments, a chemical building block may be covalently attached to the nascent member or compound in a reaction that employs multiple rounds of reagent addition and washing. For example, the solid support may be washed in order to remove the reaction mixture, and a new reaction mixture could be added, for example comprising fresh solvent and reagents. This may be useful for example in driving the reaction of the chemical building block and the nascent member towards completion, increasing the incorporation of the chemical building block and reducing the proportion of unreacted chemical building blocks and nascent members. Multiple rounds of reaction may allow for the incorporation of chemical building blocks which normally are associated with poor reaction yields, such as N-methylated amino acids.
A chemical building block may be covalently connected to the nascent member through its proximal binding group. The distal binding group of the chemical building block may be used to connect further chemical building blocks or the cleaving group to the end of the chain in subsequent steps. During the reaction of the proximal binding group of the chemical building block with the nascent member, the distal binding group of the chemical building block may be protected for example by covalent linkage to a protecting group. Before, for example, the sequential addition of the next chemical building block in a chemical building block chain, the distal binding group may be deprotected, for example by removing the protecting group.
After every chemical building block incorporation step, molecular species of the nascent member which have failed to incorporate the chemical building block (i.e. unreacted members) may be capped. For example, a capping group may be covalently attached to the unreacted capture groups after incorporation of the first chemical building block, and unreacted distal binding groups after incorporation of further chemical building blocks, to prevent the cleaving group from attaching to the unreacted capture or distal binding group.
A capping group is a chemical group that irreversibly caps a reactive group, such as a capture group or a distal binding group described herein and prevents it from taking part in any further chemical reactions. Capping groups are used in the methods described herein to stop unreacted species from one step of a method described herein from reacting in subsequent steps of the method. In particular, capping groups prevent the attachment of the cleaving group to intermediate species. For example, in a chain of chemical building blocks, the distal binding group of the chemical building block at the end of the complete chain remains uncapped and available to react with the cleaving group. This allows the cleaving group to be selectively attached to the end of complete chains of chemical building blocks.
Suitable capping reagents may include monofunctional carboxylic acid derivative reactive groups, such as acetic anhydride, for capping amines; azides for capping alkyne reactive groups; and monofunctional amines for capping carboxylic acid reactive groups.
In some embodiments, capping may not be required. For example, in some embodiments, the distal binding group of a chemical building block (e.g. building block 1) may only be used to react with the subsequent building block (e.g. building block 2) due to the nature of the functional group. Any building blocks thereafter (e.g. building block 3, building block 4, etc.) may not comprise a functional group compatible with binding to the second last building block (e.g. building block 1). Any compounds which failed to incorporate the intermediate building block (e.g. building block 2) therefore may not incorporate any further building blocks.
The same effect as with capping may be achieved thereby.
The methods described herein may be repeated one or more times using different combinations of chemical building blocks to generate a library that comprises diverse members that display different chemical portions. For example, the number, identity and/or order of the chemical building blocks may be different in the chemical portions of different members of the library.
The chemical building blocks and coding oligonucleotides may be conveniently added to nascent library members by a split and pool procedure, as described herein. Alternatively, parallel synthesis for each library member is also possible for sufficiently small libraries.
A split and pool procedure for nucleic acid encoded chemical library synthesis may comprise the steps of;
Preferably, the chemical building blocks added to the nascent member form a linear chain of chemical building blocks attached to the scaffold at a proximal end. Compounds and members comprising a complete chain of chemical building blocks can be purified by the method for self-purification described herein. A chain of chemical building blocks is best suited for maximum self-purification of the chemical portion. For chemical building blocks to be incorporated in a chain of chemical building blocks, they must be at least bifunctional (i.e. contain at least two binding groups). The proximal and distal binding groups of a chemical building block may display different or orthogonal reactivity. An example of a solid support compound with a linear arrangement of building blocks is shown in
In other embodiments, the chemical building blocks added to the nascent member may form a branched structure attached to the scaffold. The scaffold may be at least tetrafunctional (i.e. it may comprise two or more capture groups) or one or more chemical building blocks may be at least trifunctional to allow for attachment points for two or more additional chemical building blocks to form a branched structure.
In other embodiments, the chemical building blocks added to the nascent member may form a cyclic or macrocyclic structure attached to the scaffold. This may require the cross-linking of a chemical building block with another chemical building block within the chemical portion or with the scaffold. The cyclic structure may be macrocyclic. Additional cross-linking of chemical building blocks with other chemical building blocks or the scaffold may yield chemical portions with bicyclic, macrocyclic or polycyclic structures formed by chemical building blocks attached to the scaffold. An example of a solid support compound with a cyclic and branched arrangement of building blocks formed by crosslinking chemical building block1 and chemical building block3 using an additional building block, chemical building block4 is shown in
A capping step may be performed after a synthetic step in the synthesis of the member or solid support compound. Preferably, a capping step may be performed after the addition of each chemical building block and optionally each coding oligonucleotide. Chemical capping steps may include reaction with a monofunctional moiety, or ligation with a non-extendable nucleic acid molecule, such as an oligonucleotide. Functionalised solid support, linker, scaffold, chemical building blocks, the chemical portion, the cleaving group, as well as the coding nucleic acid portion may be capped. A capping step may comprise reacting an unreacted functional group, such as a binding group or capture group with a capping reagent, to form an unreactive capped group. Suitable capping reagents include activated carboxylic acid derivatives.
An example of the selective release of complete compounds or members from the solid support is shown in
The coding nucleic acid portion may be capped through the use of coding oligonucleotides with ends that are only compatible with the immediately preceding coding oligonucleotide and cannot ligate to other coding oligonucleotides added previously to the nucleic acid portion. An example of the ‘capping’ of the coding nucleic acid portion is shown in
Preparation of the member or compound as described herein may include any reaction compatible with the encoding system and the solid support, as well as linker integrity. In some embodiments, possible reactions may include DNA-compatible reactions. Possible reactions may include amide bond formation, Suzuki coupling, Sonogashira coupling, reductive amination, and copper-catalyzed alkyne-azide cycloaddition. DNA compatible reactions are described in literature including (Malone, M. L, Paegel, B. M., ACS Comb. Sci. 2016, 18 (4), 182-187).
In some embodiments, macrocyclization reactions may be used in the reaction of the cleaving group with the linker, the installation of the second linker, the crosslinking of other chemical entities in the solid support compound, or during additional transformations in solution after release from solid support. For example, in some embodiments, the second linker may first be attached to the solid support, and then be connected to the chemical portion in a macrocyclization reaction. Macrocyclization reactions may preferably include reactions which display a high yield, such as copper-catalyzed azide-alykne cyloaddition, for example. Macrocyclization reactions are known in the art and may include reactions described in (Wang, W., Khojasteh, S. C. & Su, D., Mol. (2021); Zhang, R. Y., Thapa, P., Espiritu, M. J., Menon, V. & Bingham, J. P., Bioorg. Med. Chem. (2018)).
A member or compound may be further transformed during and after the preparation. For example, a method may comprise;
A member or compound may be recaptured onto a new solid support.
In some embodiments, covalent bond formation may be mediated by a nucleic acid templated reaction.
In some embodiments, a macromolecule capable of mediating a transformation, such as an enzyme, may be employed in or after the preparation of a self-purified compound. For example, an enzyme may be recruited by a nucleic acid templated reaction.
A member of a nucleic acid encoded library comprises a nucleic acid portion.
The coding nucleic acid portion may consist of a single-stranded or double-stranded nucleic acid, or a combination of single-stranded and double-stranded nucleic acid. In some embodiments, only one nucleic acid strand may be linked to the scaffold. In other embodiments, both nucleic acid strands of a double-stranded coding nucleic acid portion may be linked to the scaffold.
The scaffold, chemical portion, and/or cleaving group may be encoded by coding sequences in the one or more nucleic acid strands of the nucleic acid portion. Elongation of the coding nucleic acid portion to incorporate a coding sequence may be performed by enzymatic ligation of a coding oligonucleotide; chemical ligation of a coding oligonucleotide; elongation across a coding oligonucleotide template using a polymerase enzyme; or a combination of any of these three methods.
A synthetic step of producing or transforming the scaffold, chemical portion, or cleaving group may be preceded or followed by addition of coding oligonucleotides to the nucleic acid portion. Coding sequences may be present on only one nucleic acid strand, or may be present on two nucleic acid strands. Coding sequences on one nucleic acid strand may be conveniently amplified, for example by PCR. Coding sequences may be on one or two nucleic acid strands and may be transcribed onto one nucleic acid strand which can be PCR amplified. A coding sequence may encode the scaffold, one or more building blocks, a cleaving group, one or more linkers or a combination thereof.
A synthetic step of producing, extending or transforming the scaffold, chemical portion, or cleaving group may preceded or followed by addition of coding sequences to the coding nucleic acid portion.
In some embodiments, only chemical building blocks may be encoded. In other embodiments, the scaffold, linker, cleaving group, or other entities may also be encoded. Although chemical building blocks which are encoded are described below, it is to be understood that also other entities may be encoded in the same way that the below described chemical building blocks are encoded.
Preferably, a member of a nucleic acid encoded library comprises a coding nucleic acid portion that encodes all of the chemical building blocks in the chemical portion that is attached to the member. Sequencing of the coding nucleic acid portion attached to a member allows the identification of the chemical building blocks in the chemical portion that is displayed by the member.
Nascent members or compounds may comprise an attachment oligonucleotide (also referred to as a headpiece). In some preferred embodiments, the attachment oligonucleotide is attached to the scaffold of the nascent member.
The attachment oligonucleotide is a nucleic acid to which coding oligonucleotides encoding chemical building blocks are attached to form the nucleic acid portion. The attachment oligonucleotide may have the same nucleotide sequence in different members of the library (i.e. a constant nucleotide sequence). The combination of coding oligonucleotides and hence the sequence of the coding nucleic acid portion may be different in different members of the library.
The attachment oligonucleotide may have an end that is attached to the nascent binding member and a free end to which coding oligonucleotides are attached. The free end of the attachment oligonucleotide may be compatible with the attachment of coding oligonucleotides. For example, the free end may comprise a short 5′ or 3′ overhang (a “sticky end”) to facilitate ligation.
The attachment oligonucleotide may be a natural nucleic acid, such as DNA or RNA, or it may be a nucleic acid analogue, such as a peptide nucleic acid (PNA), a phosphorodiamidate morpholino oligomer (PMO), a phosphorothioate oligomer (PTO), a locked nucleic acid (LNA), a glycol nucleic acid (GNA) or a threose nucleic acid (TNA).
A first coding oligonucleotide encoding the first chemical building block may be attached to the attachment oligonucleotide. Suitable techniques for attachment of oligonucleotides are well-established and include enzymatic ligation. Subsequent coding oligonucleotides encoding the second and further chemical building blocks may be attached to the previous coding oligonucleotide to form a coding nucleic acid comprising coding oligonucleotides for the chemical building blocks in the chain attached to the member.
In some embodiments, the attachment oligonucleotide may be double-stranded.
A double stranded attachment oligonucleotide may be formed from the intramolecular hybridisation of a single nucleotide strand (i.e. a hairpin) or may be formed from the intermolecular hybridisation of two separate nucleotide strands. The double stranded nucleotide sequence may be denatured to produce a single stranded nucleic acid before cleavage of the linker. Hybridisation of the single stranded nucleic acid of a first released member with the single stranded nucleic acid of a second released member may be useful for example, in producing the members of an ESAC library. Alternatively, a double stranded attachment oligonucleotide in which the two oligonucleotide strands are covalently linked may be employed.
Double-stranded coding oligonucleotides may be attached to a double stranded attachment oligonucleotide by ligation using a ligase, such as T4 DNA ligase, in accordance with standard techniques.
In other embodiments, the attachment oligonucleotide may be single-stranded. Single-stranded coding oligonucleotides may be attached to the attachment oligonucleotide by splint ligation using an adaptor oligonucleotide, in accordance with standard techniques.
A coding oligonucleotide is a nucleic acid molecule that contains a nucleotide coding sequence that encodes a chemical building block and optionally the cleaving group, scaffold and/or linker. The coding sequence (or coding region) can be any sequence of nucleic acid bases that is uniquely associated with a particular chemical building block. This allows the identity of the chemical moiety to be determined by sequencing or otherwise ‘reading’ the coding sequence.
A coding sequence contains sufficient nucleotides to uniquely identify the chemical building block for which it is coding. For example, if the chemical portion has 20 variants, the coding sequence needs to contain at least 3 nucleotides (42=16, 43=64). The coding sequence may be longer than necessary. The benefit of employing coding sequences that are longer than necessary is that they provide the opportunity to differentiate codes by more than just a single nucleotide difference, which gives more confidence in the decoding process. For example, a first chemical building block from a population of 20 different chemical building blocks (20 compounds) may be encoded by 6 nucleotides, and a second chemical building block from a population of 200 different moieties may be encoded by 8 nucleotides. The size of the coding sequence therefore depends on the number of chemical building blocks to be encoded (i.e. the number of different chemical building blocks in the library). A sequence of nucleotides and/or its complement may be used as a coding sequence to encode a chemical building block. Suitable sequences for encoding chemical building blocks in a library are well-known in the art.
The coding sequences of the coding oligonucleotides may be flanked by constant regions. The constant regions may be of sufficient length to allow an efficient hybridization and ligation, for example 2-20 bases, preferably 9-15 bases.
Coding oligonucleotides are added in a sequential fashion to the member or compound, concurrently with the incorporation of the building blocks, resulting in a nucleic acid molecule (i.e. nucleic acid portion) containing a linear series of coding oligonucleotides that encode the combination of chemical building blocks that is present in the member or compound. The first coding oligonucleotide that encodes the first chemical building block may be linked to the attachment oligonucleotide and further coding oligonucleotides may each be linked to the preceding coding oligonucleotide in the series to form a nucleic acid molecule (i.e. the nucleic acid portion). The sequence of the nucleic acid portion of a library member encodes the chemical building blocks of the library member. Sequencing the coding nucleic acid portion thus allows the chemical building blocks of a member to be identified.
The first chemical building block and coding oligonucleotide may be attached to the nascent member or compound by a method comprising;
After the reaction, unreacted species may be removed by washing or capped to prevent further reactions. For example, the method may further comprise capping scaffolds not covalently attached to the first chemical building block. Suitable methods of capping are described above.
The first chemical building block may be protected to prevent unwanted reactions. For example, the distal binding group of the first chemical building block may be covalently linked to a protecting group. The first chemical building block may be subsequently deprotected, for example after attachment of the coding oligonucleotide. A method may comprise removing the protecting group from the distal binding group of the first chemical building block attached to the scaffold. This allows the addition of a second chemical building block.
In some embodiments, the first chemical building block may be attached to the nascent compound or member by a method comprising;
Following deprotection of the distal binding group, a second chemical building block may be attached to the nascent member by a method comprising;
After the reaction, unreacted species may be removed by washing or capped to prevent further reactions. For example, the method may further comprise capping the distal binding group of any chemical building blocks not covalently attached to the further chemical building block.
The further chemical building block may be protected to prevent unwanted reactions. For example, the distal binding group of the further chemical building block may be covalently linked to a protecting group. The further chemical building block may be subsequently deprotected, for example after attachment of the coding oligonucleotide. A method may comprise removing the protecting group from the distal binding group of the further chemical building block attached to the scaffold. This allows the attachment of additional chemical building blocks or the cleaving group.
In some embodiments, a further chemical building block may be attached to the nascent member by a method comprising;
Covalent attachment of the further coding oligonucleotide may be performed before, after or at the same time as deprotection of the distal binding group.
This process may be repeated one or more times to incorporate multiple further chemical building blocks to produce the chemical portion. For example, the chemical portion may comprise a chain of 1, 2, 3, 4, 5 or more chemical building blocks. In some preferred embodiments, the chemical portion may comprise up to 20 chemical building blocks. In other preferred embodiments, the chemical portion may comprise up to 10 chemical building blocks. In other preferred embodiments, the chemical portion may comprise up to 6 chemical building blocks.
In some embodiments, the chemical building block at the end of the chain of chemical building blocks (i.e. the terminal chemical building block) may comprise a protected distal binding group, for example a distal binding group that is covalently linked to a protecting group. A method may comprise removing the protecting group from the distal binding group of the chemical building block at the end of the chain of chemical building blocks before attachment of the cleaving group.
A solid support is an insoluble body which presents a surface on which the nascent member or compound can be attached during production as described herein. Examples of suitable supports include resins, beads, nanoparticles and polymers such as polystyrene-polyethylene glycol (PEG) composites, PEG and poly-ε-lysine (ε-PL) (see for example Albericio F (2000). Solid-Phase Synthesis: A Practical Guide. Boca Raton: CRC Press). Conveniently, the support may be in form of particles, such as beads. In some embodiments, the solid support may be a bead of a grafted copolymer consisting of a polystyrene matrix grafted with poly (ethylene glycol) (PEG). Solid supports may be produced using standard techniques or obtained from commercial suppliers (e.g. Tentagel®, Rapp Polymere GmbH, DE). The separation of the compounds on solid support from a solution may be achieved by any convenient method, such as filtration, by magnetic interactions (for magnetic beads), by centrifugation, etc.
Other suitable solid supports may include polystyrene beads, crosslinked polystyrene beads, polymer beads, glass beads, coated glass beads, controlled-pore glass beads, beaded controlled-pore glass beads, silica microparticles, coated silica microparticles, iron oxide particles, coated iron oxide particles, PEGA (polyethylene glycol-acrylamide) resin, and other commercially available or custom synthesized solid supports of different sizes, or combinations thereof. Suitable solid supports may be magnetic. Examples of magnetic solid supports include Magnefy™ and ProMag 1® microspheres (Bangs Laboratories, Inc.). Examples of solid supports may include co-polymers, such as acrylamide-PEG co-poymer, polymer particles which additionally comprise a material which is paramagnetic or ferromagnetic, core-shell particles, porous particles, non-porous particles, or other organic chemical materials in combination with a ferromagnetic material. Other suitable solid supports are known in the art (see for example, Pon, R. T. Curr. Protoc. Nucleic Acid Chem. (2000); Chaudhuri, R. G. & Paria, S., Chem. Rev. (2011); Wu, W., He, Q. & Jiang, C. Nanoscale Res. Lett. (2008); Hermanson, G. T., Bioconjugate Techniques: Third Edition (2013)).
In some embodiments, a binding entity may be used for capture onto solid support. For example, a small organic or inorganic entity may be used for capture onto a solid support to allow the physical separation of compounds bond to the solid support from a solution. Suitable small organic or inorganic binding entities may include biotin and quantum dots, such as magnetic quantum dots. In some embodiments, one or more steps in the synthesis of a nucleic acid encoded compound or library as described herein may be performed in solution before or after capture onto a solid support.
In some embodiments, solid supports may be additionally functionalized by a linear (for example, polyethylene glycol (PEG) spacer) or dendrimer structure (for example, polyamidoamine (PAMAM) dendrimer). Dendrimers and spacers are described in literature including (Hermanson, G. T., Bioconjugate Techniques: Third Edition (2013)). In some embodiments, the solid support surface may be modified by a small molecule. In some embodiments, for example, the small molecule may connect a part of the solid support with the first and/or second linker.
Preferably, the collection of solid support particles can be readily suspended in a solution to allow for splitting and pooling, if this is desired. In some embodiments, a small solid support particle size may be preferable for the facile synthesis of a library with a large number of distinct members. For example, microparticles or nanoparticles may be used.
The solid support allows the bound member or compound to be washed after one or more steps of construction as described herein to remove unbound reactants. Only complete members are capable of self-release. This may allow the separation of pure library members from incompletely synthesized library members, allowing the production of DELs with high purity. In some embodiments, the separation of the released members in solution from species that remain attached to the solid support allows the enrichment or purification of complete members, allowing the production of DELs with high purity. In other embodiments, the separation of the released members in solution is preceded by a step which may only or preferentially release incompletely synthesized species from the solid support. The subsequent release of members that remain attached to the solid support allows the enrichment or purification of complete members, allowing the production of DELs with high purity.
In some embodiments, members or compounds as described herein are released from the solid support by the reaction of the cleaving group and the linker. An electrophilic cleaving group and a nucleophilic linker may be employed or more preferably a nucleophilic cleaving group and an electrophilic linker. In some embodiments, the reaction between the linker and the cleaving group may be a substitution reaction, substituting the solid support with the cleaving group. The substitution reaction may be a nucleophilic substitution reaction, for example a nucleophilic aromatic substitution reaction. Other suitable reactions between the linker and the cleaving group may include metal-catalysed reactions or metathesis reactions.
In some preferred embodiments, one of the linker and the cleaving group may be a thiol or selenothiol group and the other may be a carbonyl group. This reaction may result in the formation of a thioester intermediate. The thioester intermediate may subsequently be cleaved intermolecularly or intramolecularly, which may result in the irreversible cyclisation of the library member (
A linker is a cleavable chemical moiety that may, in some embodiments, connect the scaffold to the solid support. The linker may connect the scaffold directly to the solid support or indirectly, for example through an anchor. The linker may be cleaved through a chemical reaction mediated by specific reagents (e.g. cleaving group) or reaction conditions.
In other embodiments, a first and a second linker may be present. The first linker may be a cleavable chemical moiety that covalently connects the scaffold to the solid support. The second linker may be a cleavable chemical moiety that covalently connects the chemical portion to the solid support. The first and second linkers may be orthogonally cleavable i.e. the first linker may be cleaved by specific reagents (e.g. cleaving group) or reaction conditions that do not cleave the second linker.
In some embodiments, a linker may not require further transformation or activation after attachment to the solid support and the scaffold before reaction with the cleaving group. The linker may be incorporated into the nascent member in an active state (i.e. the linker is in a form that is reactive to the cleaving group). Suitable linkers include substituted quinoxalines or derivatives thereof, which may be cleaved by an ortho-dithiophenol cleaving group without further transformation or activation.
A substituted-quinoxaline may comprise a quinoxaline group with one or more substitutions, for example substitutions at positions 2, 3 and 5 or positions 2, 3 and 6. In some embodiments, position 2 of a substituted quinoxaline may be a halogen, such as F, Cl, Br or I, preferably CI, or an electron withdrawing group; position 3 may be —SR, —OR or —NR and position 5 or 6 may be —COOR, —CONR, or alkyne. In other embodiments, position 2 of a substituted quinoxaline may be —SR, —OR or —NR; position 3 may be a halogen, such as F, Cl, Br or I, preferably CI, or an electron withdrawing group and position 5 or 6 may be —COOR or —CONR, or alkyne. R may be independently selected from a hydrogen atom, or a C1-6 alkyl group, C6-20 aryl, a C1-6 alkoxy group, a C1-6 acyloxy group, or a C1-6 reverse ester group; any of which may be linear or branched and optionally substituted. Examples of suitable substituted-quinoxalines may include 3-chloro-2-((2-hydroxyethyl)thio)quinoxaline-6-carboxylic acid; 3-chloro-2-(4-(hydroxymethyl)phenoxy)quinoxaline-6-carboxylic acid; and N-(3-aminopropyl)-3-chloro-2-(4-(hydroxymethyl)phenoxy)quinoxaline-6-carboxamide.
In other embodiments, the linker may require activation after attachment to the solid support and the scaffold and before reaction with the cleaving group i.e. the linker may be an activatable linker. The linker may be incorporated into the nascent member in an inactive state, (i.e. the linker is in a form that is not reactive to the cleaving group). Activation of the linker converts it from a non-activated form into an activated form. The activated form of the linker is selectively cleaved by the cleaving group, whereas the non-activated form of the linker is not cleaved by the cleaving group. For example, the inactive form of the linker may comprise a protecting group and the linker may be activated by removal of the protecting group. A method described herein may comprise activating the activatable linker. In embodiments in which the cleaving group requires activation, the linker may be activated before, after, or simultaneously with the activation of the cleaving group.
Suitable activatable linkers include masked thioesters, such as N-alkyl cysteine. Masked thioesters may be activated to produce a thioester that may be cleaved by a thiol cleaving group (native chemical ligation). The thiol in the masked thioester may for example be protected by a tert-butyl group, allyloxycarbonylaminomethyl group, 2-nitroveratryl group, 9-fluorenylmethyl group, or as an S-sulfonate. After deprotection of the masked thioester, the cysteine derivative may undergo an N to S rearrangement upon deprotection of the thiol to give a thioester.
Other suitable activatable linkers include diaminobenzoyl groups or derivatives thereof, such as methyl diaminobenzoyl groups. For example, the activatable linker may be amino (methyl) aniline (MeDbz). MeDbz may be activated by reaction with para-nitrophenyl choloroformate to produce N-acyl N′-methyl benzimidazoline (MeNbz), which may be cleaved by a thiol cleaving group. Other examples of suitable activatable linkers include 3,4-diaminobenzoic acid (Dbz), and derivatives thereof, which may be activated with isopentyl nitrite to produce a benzotriazole derivative (Selvaraj, A. et al, Chem. Sci., 2018, 9, 345-349).
Other suitable activatable linkers include enzyme substrates. For example, the activated linker may be an oligonucleotide that is cleaved by a nuclease cleaving group or a peptide that is cleaved by a peptidase.
In some embodiments, a linker is cleaved by a cleaving group to release the solid support compound. The cleaving group is a reactive chemical group, reagent, or enzyme that is capable of reacting with the linker to cleave the linker and release the nascent member from the solid support. In order to provide self-purified compound, the cleaving group may be either covalently linked to or reversibly associated with the chemical portion, the nucleic acid portion, or the scaffold. For example, the cleaving group may be attached to the distal end of a chain of chemical building blocks after completion of the chemical portion. The cleaving group may be attached to the distal binding group of the chemical building block at the distal end position of the chain of chemical building blocks in the chemical portion. In other embodiments, the cleaving group may be attached to any position in the chemical portion, to the scaffold, or to the nucleic acid portion.
In some embodiments, the cleaving group does not require further transformation or activation after attachment to the chemical portion, scaffold, or the coding nucleic acid portion and before reaction with the linker.
In other embodiments, the cleaving group requires activation after attachment to the chemical portion, scaffold, or the coding nucleic acid portion and before reaction with the linker i.e. the cleaving group may be an activatable cleaving group. The cleaving group may contain a functional group which is protected upon integration of the cleaving group into the member or compound, and which after deprotection cleaves the linker. For example, the cleaving group may comprise a protecting group and may be activated by removing the protecting group.
In some preferred embodiments, the cleaving group may be protected. For example, it may be covalently linked to a protecting group. A protected cleaving group may be inactive and may be activated by deprotection. A method may further comprise deprotecting the cleaving group, for example by removing the protecting group. The cleaving group may be deprotected before, after or simultaneously with a potential activation of the linker. Suitable protecting groups are described above.
A cleaving group may preferably be at the end of the chain of chemical building blocks that form the chemical portion attached to the scaffold (i.e. the distal end of the chemical portion). Alternatively, a cleaving group may be attached to a chemical building block within the chain other than the terminal chemical building block or incorporated between two chemical building blocks in the chain.
In some embodiments, a compound or member may comprise multiple different cleaving groups. For example, two different or orthogonal cleaving groups may be attached to two different positions in the chemical portion.
The choice of cleaving group will depend on the activatable linker.
Suitable cleaving groups may comprise or consist of a thiol. In some embodiments, a thiol cleaving group may be used to cleave a thioester linker, for example, a thioester linker produced by activation of a masked thioester, or masked N-alkyl cysteine; or a diaminobenzoyl linker such as the MeNbz linker, for example a MeNbz linker produced by activation of MeDbz. The thiol may be protected during the incorporation into the solid support compound.
Other suitable cleaving groups may comprise or consist of a selenothiol, which may be protected during incorporation into the member or compound on the solid support. The selenothiol may display similar reactivity with a linker compared to a thiol. The selenothiol may be protected during the incorporation into the solid support compound. For example, the cleaving group may be cysteine or a derivative thereof, or selenocysteine, or a derivative thereof. An amine in the cleaving group in addition to a thiol or selenothiol may result in intramolecular cleavage of the thioester or selenoester formed after reaction of the cleaving group with the linker by the amine.
In some embodiments, the cleaving group may comprise multiple thiol or selenothiol groups.
Other suitable cleaving groups may comprise or consist of an ortho-dithiophenol. An ortho-dithiophenol cleaving group may be used to cleave a substituted quinoxaline linker. Substituted quinoxaline linkers are described in more detail above.
Other suitable cleaving groups may comprise or consist of an enzyme. An enzyme cleaving group may be used to cleave a linker comprising an enzymatically cleavable structure. For example, a nuclease cleaving group may be used to cleave a polynucleotide linker and a peptidase may be used to cleave peptide linker.
Other reactions may be used for the cleavage of the linker by the cleaving group. Reactions which are compatible with the selective reaction of a cleaving group and a linker as described herein may be employed. For example, the linker may be an N- and O-substituted hydroxylamine, and the cleaving group may comprise an alpha-ketoacid group. In another example, the linker may comprise a carboxylic acid ortho-hydroxybenzaldehyde ester, which is further derivatized on the aromatic ring, and the cleaving group may comprise serine, or a derivative thereof, or threonine, or a derivative thereof, linked to the chemical portion, the scaffold, or the coding nucleic acid portion via its carboxyl group.
The same or different types of protecting groups may be used for the scaffold, chemical building blocks and the cleaving group, as long as they do not undesirably interfere in the synthesis of a self-purified compound or member.
For example, a thiol cleaving group may be protected by covalent linkage to an allyloxycarbonyl-aminomethyl group, o-nitrobenzyl group, 2-nitroveratryl (Nv) group, tert-butyl (tBu) group, 9-fluorenylmethyl group, through formation of a S-sulfonate, or through a disulfide bond or selensulfide bond. Preferably, the protection through a disulfide or selensulfide bond may be intramolecular.
A selenothiol cleaving group may be protected through a selensulfide or a diselenide bond.
An ortho-dithiophenol cleaving group may be protected by covalent linkage by intermolecular disulfide bond formation of each thiol. For example, thiol groups in the ortho-dithiophenol cleaving group may each be protected by a S-tert-butyl group. In some embodiments, the thiols may be protected by other protecting groups mentioned above.
In some embodiments, the protecting group of a functionality in the cleaving group may be photolabile and may be attached to the cleaving group by a photolabile bond. The protecting group may be removed by the application of light to cleave the photolabile bond and activate the cleaving group. The cleaving group may be deprotected before, after or simultaneously with a potential activation of the linker. Suitable photolabile protecting groups include 2-nitrobenzyl groups, such as the 2-nitroveratryl group.
In other embodiments, the solid support member may comprise first and second linker that are independently cleavable by exposing the member to suitable conditions, without the requirement for a cleaving group. The first linker may connect the scaffold to the solid support. The second linker may connect the chemical portion to the solid support. Cleavage of the first linker may release members that are not also connected through a second linker (i.e. members with an incomplete chemical portion).
One enzyme or different enzymes may mediate one reaction or multiple reactions involved in the production of a self-purified compound or member as described herein.
Selectively released self-purified library members may comprise a linear, branched, cyclic, macrocyclic, or polycyclic structure formed from the chemical portion and optionally the scaffold, and one or more cleavage moieties resulting from the reaction of the linker and the cleaving group, or one or more moieties remaining after cleavage of linkers.
After self-purified library members are selectively released from solid support, further reactions may be performed. For example, additional transformations may be performed in solution, or the self-purified compound may be re-captured onto solid support for additional transformations including transformations followed by self-purification reactions. Further transformations may include chemical building block incorporation reactions, crosslinking of chemical building blocks, crosslinking of chemical building blocks to the scaffold, cleavage reactions, ring-opening reactions, and macrocyclization reactions. For example, a bicyclic structure may be formed after a macrocyclic self-purified library member is released from solid support by crosslinking of two building blocks in a CuAAC (copper-catalysed azide-alkyne cycloaddition).
In some embodiments, individual solid support-linked library members may be compartmentalised before the member is released from the solid support. This allows for activity-based assays of self-purified library members. This also allows the released member to be physically separated from the nucleic acid molecule comprising the coding oligonucleotides within the compartment. The presence of the nucleic acid molecule and the released member in the same compartment allows the nucleic acid molecule encoding the released member to be identified and sequenced to determine the chemical building blocks that form the chemical portion of the member. This may be useful for example in activity-based assay systems.
Members may be compartmentalised by isolating each particle of the solid support in a separate compartment. Segregation prevents solid support-bound members of different particles from interacting with each other and allows the coding nucleic acid to remain associated with the released member, even it when it is physically separated from it by the cleavage of the linker. A compartment may comprise an isolated volume or droplet. For example, a volume or droplet of between about 0.5 pL and about 100 nL may be used. However, smaller or larger volumes may also be used. The compartment may comprise the solid support-bound member and suitable reactants, buffers and other reagents to facilitate cleavage of the linker and release of the member from the support. The compartmentalized library may be in any suitable format, for example in an array, microfluidic or micropatterned device, or multiwell dish.
In some embodiments, the coding nucleic acid portion comprising the coding oligonucleotides may be attached to an anchor located between the linker and the solid support, such that the anchor and the coding nucleic acid portion remain linked to the solid support when the linker is cleaved and the member released.
The anchor is a chemical moiety to which the attachment oligonucleotide is attached. Preferably, the same chemical moiety forms the anchor for all of the members of the library.
In some embodiments, a method of producing a DNA encoded library may comprise for each member the steps of;
In other embodiments, a method of producing a DNA encoded library may comprise for each member the steps of;
The chemical portion remains compartmentally associated with the nucleic acid portion, allowing the sequencing to the nucleic portion to identify the compartmentally associated chemical portion.
In other embodiments, the coding nucleic acid portion comprising the coding oligonucleotides may be attached to the solid support, such that the coding nucleic acid portion remains linked to the solid support when the linker is cleaved and the member released. For example, a method of producing a DNA encoded library may comprise for each member the steps of;
Another method of producing a DNA encoded library may comprise for each member the steps of;
In some preferred embodiments of the above aspects of the invention, between 1 and 1015 copies of the linker may be present on one solid support entity, such as a particle or bead.
Following release from the support, the member or compound may be additionally purified. For example, additional HPLC purification may be performed after self-purification.
A library produced by a method described herein may be screened for members that bind to a target molecule. Library members that bind to the target molecule may be identified and the nucleic acid molecules sequenced to identify the chemical building blocks that form the chemical entities displayed by the identified library members.
The binding of identified library members may be validated in the absence of coding nucleic acid. For example, a method may comprise;
The binding of the released member to the target molecule may be determined using the label. The scaffold may be labelled with any convenient label, for example a fluorescent label.
Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of” and the aspects and embodiments described above with the term “comprising” replaced by the term “consisting essentially of”.
It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise.
Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention.
All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.
Priority is claimed from EP20203475.7, the disclosure of which is also incorporated herein by reference in its entirety for all purposes.
“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.
Experiments
Materials and Methods
Resin Swelling
Before first use or after storage at −20° C., TentaGel® beads (Rapp Polymere GmbH) were incubated with a suitable solvent for 10 min.
Solid Support Drying and Washing
TentaGel® beads (Rapp Polymere GmbH) were used in a solid-phase synthesis reaction vessel with a frit. Vacuum filtration was used to remove any solvent or solution incubated with the TentaGel® beads. The beads were washed by adding a suitable solvent, and subsequently removing the resulting solvent or solution. Magnetic solid support was separated from solutions by using a magnet.
Solid Support Quantities
The quantity of solid support used in each experiment is given in mmol of the respective surface functionality (loading amount), in mass (g or mg), or as a volume (μL), wherein the volume of beads refers to the volume of bead suspension used at the same concentration as supplied by the manufacturer.
Solid Support Storage
Functionalized TentaGel® beads (Rapp Polymere GmbH) were stored dried at −20° C. Other solid support types were stored as a suspension in a suitable solvent or aqueous solution at 4° C.
Oligonucleotides
Custom oligonucleotides purchased were additionally purified by ethanol precipitation. The redissolved oligonucleotides in mQ were analysed by LCMS and their concentration was measured using a NanoDrop 2000c Spectrophotometer.
Ethanol Precipitation of Oligonucleotides
Ethanol precipitation was performed by adding 10% (v/v) 5 M NaCl and 3.5 volumes of EtOH. The samples were stored at −20° C. for at least 2 h, and then centrifuged at 4° C. for 1 h at 20800×g. The supernatants were discarded following centrifugation. The precipitate was completely dried in a Christ Alpha RVC Speedvac rotational vacuum concentrator instrument, and then dissolved in mQ (Milli-Q®) water.
Concentration Assessment of Oligonucleotide Solutions
The concentration of oligonucleotide solutions was determined using a NanoDrop 2000c Spectrophotometer instrument by the measurement of UV absorbance at 260 nm. 2 μL of the oligonucleotide solution was used for each measurement. The concentration of the oligonucleotide was calculated from the known absorption coefficient of the DNA sequence and the measured UV absorbance at 260 nm.
Liquid Chromatography-Mass Spectrometry (LCMS)
Mass spectrometry (LCMS) spectra were recorded using a Waters Acquity UPLC and Xevo G2-XS QTof Quadrupole Time of Flight Mass Spectrometer (Waters). An XBridge® Oligonucleotide BEH™ C18, 130 Å, 2.5 μm, 2.1 mm×50 mm column or an XBridge® Oligonucleotide BEH™ C18, 130 Å, 1.7 μm, 2.1 mm×50 mm column was used for LCMS analysis of oligonucleotides. An Acquity UPLC® CSH™ C18, 1.7 μm, 2.1 mm×50 mm column was used for LCMS analysis of small molecules.
General Procedure 1: General Procedure for Amide Coupling of a Carboxylic Acid to Amine-Functionalized Solid Support
The functionalized TentaGel® beads were swollen in DMF (10 mL). The amine-functionalized TentaGel® beads (1 equiv. amine loading amount) were incubated with 66.7 mM respective acid (4 equiv.), 133.3 mM N,N-diisopropylethylamine (DIPEA) (8 equiv.), and 66.7 mM N-[(Dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equiv.) in dimethylformamide (6 mL per 0.1 mmol loading amount) at room temperature on a rotational shaker for 2 h. The functionalized TentaGel® beads were washed with dimethylformamide (3×10 mL), dichloromethane (3×10 mL), and then dimethylformamide (3×10 mL).
General Procedure 2: General Procedure for Fmoc Deprotection on Solid Support
The functionalized TentaGel® beads were swollen in DMF (10 mL). The dried functionalized TentaGel® beads were then incubated with 20% (v/v) piperidine in dimethylformamide (6 mL per 0.1 mmol loading amount) at room temperature on a rotational shaker for 30 min. The functionalized TentaGel® beads were washed with dimethylformamide (3×10 mL), dichloromethane (3×10 mL), and then dimethylformamide (3×10 mL).
General Procedure 3: General Procedure for Amide Coupling of an Acid to Amine-Functionalized Magnetic Solid Support
Amine-functionalized solid support (50 μL) was washed with dimethylformamide (200 μL). The amine-functionalized beads were incubated with a solution of 50 mM diisopropylcarbodiimide (DIC), 50 mM ethyl cyano(hydroxyimino)acetate (OxymaPure) and 50 mM respective acid in dimethylformamide (150 μL) on a rotational shaker at room temperature for 4 h. The functionalized beads were washed with dimethylformamide (6×200 μL).
General Procedure 4: General Procedure for Fmoc Deprotection on Magnetic Solid Support
The functionalized beads (50 μL) were washed with dimethylformamide (200 μL). The functionalized beads were incubated with 20% (v/v) piperidine in dimethylformamide (200 μL) at room temperature on a rotational shaker for 1 h. The functionalized beads were washed with dimethylformamide (6×200 μL).
General Procedure 5: Attachment of 5′-Azido Modified Single-Stranded Oligonucleotide (or 5′-Azido Modified Double-Stranded Oligonucleotide) to Solid Support by CuAAC Method 1
The procedure was adapted from MacConnell et al 2015. Alkyne-functionalized TentaGel® beads (20 mg) were swollen in 30 mM triethylammonium acetate pH 8.5 in 50% DMSO in mQ water with 0.034% (v/v) Tween 20 on a rotational shaker at room temperature for 15 min. The dried solid support was reacted with 1-5 nmol 5′-azido modified single-stranded oligonucleotide (synthesized in Example 1) in a solution of 2.6 mM 1-(Phenylmethyl)-N,N-bis[[1-(phenylmethyl)-1H-1,2,3-triazol-4-yl]methyl]-1H-1,2,3-triazole-4-methanamine (TBTA), 14.0 mM sodium ascorbate, and 2.8 mM copper sulfate in 20 mM triethylammonium acetate pH 8.5 in 48% DMSO in mQ water with 0.035% (v/v) Tween 20 (195 μL) on a rotational shaker at 60° C. for 3 h. The solid support was washed with a solution of 10 mM 2,2′-(1,3-Propanediyldiimino)bis[2-(hydroxymethyl)-1,3-propanediol], 100 mM sodium chloride, 10 mM N,N′-1,2-Ethanediylbis[N-(carboxymethyl)glycine] (EDTA) with 1% (v/v) Tween 20 and 1% (w/v) sodium dodecyl sulfate (SDS), pH 7.6 (3×0.6 mL).
General Procedure 6: Attachment of 5′-Azido Modified Single-Stranded Oligonucleotide to Solid Support by CuAAC Method 2
Alkyne-functionalized magnetic solid support (25 μL) with an excess loading capacity was washed with 50% DMSO in mQ water (3×200 μL). The solid support was reacted with 1-5 nmol 5′-azido modified single-stranded oligonucleotide (Synthesized in Example 1) in a mixture of 993 μM 1-(Phenylmethyl)-N,N-bis[[1-(phenylmethyl)-1H-1,2,3-triazol-4-yl]methyl]-1H-1,2,3-triazole-4-methanamine (TBTA), 945 μM copper sulfate (CuSO4), and 5.672 mM sodium ascorbate (NaAsc), and 100 mM lithium chloride in 42% DMSO in mQ water (90 μL) on a rotational shaker at room temperature for 1 h. The solid support was washed with 50% DMSO in mQ water (3×200 μL).
Amino-modified ssDNA: 5′ d Amino C6-GGAGCTTCTGAATT 3′ (Sequence 1)
Acid azide: 3-[2-[2-[2-(2-Azidoethoxy)ethoxy]ethoxy]ethoxy]propanoic acid
3-[2-[2-[2-(2-Azidoethoxy)ethoxy]ethoxy]ethoxy]propanoic acid (300 μL, 100 mM in DMSO), 1-Hydroxy-2,5-dioxo-3-pyrrolidinesulfonic acid (S—NHS) (145 μL, 100 mM in 33% (v/v) mQ water in DMSO), and N3-(Ethylcarbonimidoyl)-N1,N1-dimethyl-1,3-propanediamine (EDC) (145 μL, 100 mM in DMSO) were added to a tube containing 1 mL dimethylsulfoxide (DMSO) and were incubated on a rotational shaker at 37° C. for 30 min. In the meantime, 250 nmol amino-modified ssDNA in 504 μL mQ water was incubated with 350 μL 250 mM borate buffer pH 9.5 on a rotational shaker at 37° C. for 30 min. The activation solution containing S—NHS, EDC, and 3-[2-[2-[2-(2-Azidoethoxy)ethoxy]ethoxy]ethoxy]propanoic acid was combined with the DNA in borate buffer and left to react for 45 min at 37° C. on a rotational shaker. The reaction was monitored by LCMS. The DNA was precipitated by addition of 20% (v/v) of 5 M NaCl followed by 3.5 volumes of absolute ethanol. The sample was stored at −20° C. for 18 h, and was then centrifuged at 4° C. and 4000×g. The supernatant was discarded, and the precipitate was completely dried in a Speedvac rotational vacuum concentrator instrument. The dried precipitate was dissolved in 1 mL 100 mM TEAA pH 7.0. The crude was purified by RP-HPLC using a Waters XBridge® BEH C18 OBD™ Prep Column (130 Å, 5 μm, 10 mm×150 mm) and a gradient of buffer 1, 100 mM TEAA pH 7.0 buffer, and buffer 2, 100 mM TEAA pH 7.0 in 80% acetonitrile in mQ water. The collected fractions were combined and concentrated, and the oligonucleotide was precipitated by addition of 20% (v/v) of 5 M NaCl followed by 3.5 volumes of absolute ethanol. The sample was stored at −20° C. for 2 h, and was then centrifuged at 4° C. and 4000×g. The supernatant was discarded, and the precipitate was completely dried in a Speedvac rotational vacuum concentrator instrument. The dried precipitate was dissolved in 500 μL mQ water. Concentration assessment by measurement of UV Absorbance at 260 nm on a NanoDrop 2000c spectrophotometer showed a 61% yield. The product was analyzed by LCMS (
NH2-functionalized 10 μm TentaGel® beads (Rapp Polymere GmbH) (385 mg, 0.1 mmol loading amount) were added to a solid-phase synthesis reaction vessel with a frit. The TentaGel® beads were swollen with dimethylformamide (6 mL) at room temperature for 10 min on a rotational shaker, and then washed with dimethylformamide (3×10 mL).
Step 2.1 Coupling to Mono-Tert-Butyl Succinate
The functionalized TentaGel® beads were coupled to mono-tert-butyl succinate following general procedure 1.
Step 2.2 Capping
The functionalized TentaGel® beads were swollen with dichloromethane (6 mL) at room temperature for 10 min on a rotational shaker, and then washed with dichloromethane (3×10 mL). The functionalized TentaGel® beads were incubated with a mixture of dichloromethane (6 mL), acetic anhydride (2 mL), and N,N-diisopropylethylamine (DIPEA) (2 mL) at room temperature for 1 h. The functionalized TentaGel® beads were washed with DCM (3×10 mL).
Step 2.3 Tert-Butyl Deprotection
To the functionalized TentaGel® beads was added a mixture of 50% (v/v) trifluoroacetic acid in dichloromethane (5 mL). After 10 min on the rotational shaker at room temperature, the solution was removed. Then, a fresh mixture of 50% (v/v) trifluoroacetic acid in dichloromethane (5 mL) was incubated with the TentaGel® beads for 20 min at room temperature on the rotational shaker. The resin was washed with dichloromethane (3×10 mL), and then dimethylformamide (3×10 mL).
Step 2.4 Coupling to N-Boc-ethylenediamine
The functionalized TentaGel® beads were swollen in DMF (10 mL). The dried functionalized TentaGel® beads were then incubated with N-Boc-ethylenediamine (63 μL, 0.4 mmol, 4 equiv.), N,N-diisopropylethylamine (DIPEA) (136 μL, 0.8 mmol, 8 equiv.), and N-RDimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethyleneFN-methylmethanaminium hexafluorophosphate N-oxide (HATU) (152 mg, 0.4 mmol, 4 equiv.) in dimethylformamide (6 mL) at room temperature on a rotational shaker for 2 h. The functionalized TentaGel® beads were washed with dimethylformamide (3×10 mL), dichloromethane (3×10 mL), and then dimethylformamide (3×10 mL).
Step 2.5 Boc Deprotection
To the functionalized TentaGel® beads was added a mixture of 50% (v/v) trifluoroacetic acid in dichloromethane (5 mL). After 10 min on the rotational shaker at room temperature, the solution was removed. Then, a fresh mixture of 50% (v/v) trifluoroacetic acid in dichloromethane (5 mL) was incubated with the TentaGel® beads for 20 min at room temperature on the rotational shaker. The resin was washed with dichloromethane (3×10 mL), and then dimethylformamide (3×10 mL).
Step 2.6 Coupling to 6-(Fmoc-amino)hexanoic Acid
The functionalized TentaGel® beads were swollen in DMF (10 mL). The functionalized TentaGel® beads were then coupled to 6-(Fmoc-amino)hexanoic acid following general procedure 1.
Step 2.7 Fmoc Deprotection
The functionalized TentaGel® beads were Fmoc deprotected according to general procedure 2.
Step 2.8 Coupling to 3-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-4-(methylamino)benzoic Acid
The functionalized TentaGel® beads were reacted with 3-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-4-(methylamino)benzoic acid (Fmoc-MeDbz-OH) following general procedure 1.
Step 2.9 Fmoc Deprotection
The functionalized TentaGel® beads were Fmoc deprotected according to general procedure 2.
Step 2.10 Coupling to 6-(Fmoc-amino)hexanoic Acid
The functionalized TentaGel® beads were reacted with 6-(Fmoc-amino)hexanoic acid following general procedure 1.
Step 2.11 Fmoc Deprotection
The functionalized TentaGel® beads were Fmoc deprotected according to general procedure 2.
Step 2.12 Coupling to (2S)-2-[[(9H-Fluoren-9-ylmethoxy)carbony]amino]-4-pentynoic Acid
The functionalized TentaGel® beads were coupled to (2S)-2-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-4-pentynoic acid (Fmoc-Pra-OH) following general procedure 1.
Step 2.13 Fmoc Deprotection
The functionalized TentaGel® beads were Fmoc deprotected according to general procedure 2.
Step 2.14 Oligonucleotide Attachment
The compound synthesized in steps 2.1-2.13 is solid support with a MeDbz linker connecting to the scaffold, which comprises a site for building block attachment at the amine functional group, and an alkyne for nucleic acid attachment. 5 nmol 5′-azido modified single-stranded oligonucleotide (synthesized in Example 1) was attached to functionalized TentaGel (steps 2.1-2.13, 20 mg) following general procedure 5.
Nascent Library Member Cleavage for LCMS Analysis
Step 2.15 MeDbz Linker Activation Step 1—Incubation with p-Nitrophenyl Chloroformate
The functionalized TentaGel® beads (20 mg) with oligonucleotide attached were incubated with 100 mM p-nitrophenyl chloroformate in dichloromethane (600 μL) on a rotational shaker at room temperature for 30 min. The resin was washed with dichloromethane (3×600 μL).
Step 2.16 MeDbz Linker Activation Step 2—Incubation with N,N-diisopropylethylamine (DIPEA)
The functionalized TentaGel® beads (20 mg) with oligonucleotide attached were incubated with 8.7% (v/v) N,N-diisopropylethylamine (DIPEA) in dichloromethane (600 μL) on a rotational shaker at room temperature for 30 min. The resin was washed with dichloromethane (2×600 μL).
Step 2.17 MeDbz Cleavage by Cysteamine
The functionalized TentaGel® beads (20 mg) with oligonucleotide attached were incubated with excess cysteamine in 50% (v/v) DMSO in mQ water with 0.01% (w/v) sodium dodecyl sulfate (SDS) (150 μL) on a rotational shaker at 60° C. for 1 h. The resulting solution was separated from the beads by centrifugation and was collected. The collected solution was analyzed by LCMS (
The functionalized solid support prepared in steps 2.1-2.13 was used as a starting material for the synthesis of the self-elution model compound off-DNA. The functionalized TentaGel® beads were swollen with dimethylformamide (6 mL) at room temperature for 10 min on a rotational shaker, and then washed with dimethylformamide (3×10 mL).
Step 3.1 Resin Splitting
The functionalized TentaGel® beads prepared in steps 2.1-2.13 were split into two portions of 0.05 mmol. The synthesis was continued on a 0.05 mmol scale with one of the two portions.
Step 3.2 Coupling to 6-(Fmoc-amino)hexanoic Acid
The functionalized TentaGel® beads were reacted with 6-(Fmoc-amino)hexanoic acid following general procedure 1.
Step 3.3 Fmoc Deprotection
The functionalized TentaGel® beads were Fmoc deprotected according to general procedure 2.
Step 3.4 Coupling to (2S)-2-([[(9H-Fluoren-9-yl)methoxy]carbonyl]amino)-3-(naphthalen-1-yl)propanoic Acid
The functionalized TentaGel® beads were reacted with (2S)-2-([[(9H-Fluoren-9-yl)methoxy]carbonyl]amino)-3-(naphthalen-1-yl)propanoic acid (Fmoc-1-NaI—OH) following general procedure 1.
Step 3.5 Resin Splitting
The functionalized TentaGel® beads were split into two portions of 0.025 mmol. The synthesis was continued on a 0.025 mmol scale with one of the two portions.
Step 3.6 Fmoc Deprotection
The functionalized TentaGel® beads were Fmoc deprotected according to general procedure 2.
Step 3.7 Coupling to (RS)-Lipoic Acid
The functionalized TentaGel® beads were reacted with (RS)-Lipoic acid following general procedure 1.
Step 4.1 Oligonucleotide Attachment
5 nmol 5′-azido modified single-stranded oligonucleotide was attached to the self-elution model compound synthesized in Example 3 (20 mg) following general procedure 5.
Step 4.2 MeDbz Linker Activation Step 1—Incubation with p-Nitrophenyl Chloroformate
The functionalized TentaGel® beads (20 mg) with oligonucleotide attached were incubated with 100 mM p-nitrophenyl chloroformate in dichloromethane (600 μL) on a rotational shaker at room temperature for 30 min. The resin was washed with dichloromethane (3×600 μL).
Step 4.3 MeDbz Linker Activation Step 2—Incubation with N,N-diisopropylethylamine (DIPEA)
The functionalized beads were incubated with 8.7% (v/v) N,N-diisopropylethylamine (DIPEA) in dichloromethane (600 μL) on a rotational shaker at room temperature for 30 min. The resin was washed with dichloromethane (3×600 μL).
Step 4.4 Cleaving Group Deprotection
The functionalized beads were incubated in a solution of 100 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in 6% (v/v) triethylamine, 53% dimethylsulfoxide (DMSO) in mQ water with 0.01% (w/v) sodium dodecyl sulfate (SDS), pH 8-9 (150 μL) on a rotational shaker at 60° C. for 1 h. The beads were dried by centrifugation.
Step 4.5 Self-Elution of Model Nucleic Acid Encoded Library Member
The functionalized beads were incubated with a solution of 10% (v/v) N,N-diisopropylethylamine (DIPEA) in 80% acetonitrile in mQ water with 0.01% (w/v) sodium dodecyl sulfate (SDS) (150 μL) on a rotational shaker at 60° C. for 1 h. The resulting solution was separated from the beads by centrifugation and was collected. The collected solution was concentrated using a Speedvac rotational vacuum concentrator instrument.
Step 4.6 Ethanol Precipitation of Self-Eluted Model Nucleic Acid Encoded Library Member
The residue obtained after step 4.5 was resuspended in 50% (v/v) dimethylsulfoxide (DMSO) in mQ water (150 μL). The sample was filtered and 10 μL were of the sample was used for LCMS analysis. 125 μL of the sample was used to ethanol precipitate the self-eluted model nucleic acid encoded library member by adding 10% (v/v) of 5 M sodium chloride (12.5 μL), 10% (v/v) of 2.5 M sodium acetate buffer pH 4.79 (12.5 μL), followed by 3.5 volumes of absolute ethanol (525 μL). The sample was stored at −20° C. for 18 h, and was then centrifuged at 4° C. and 20800×g for 1 h. The supernatant was discarded, and the precipitate was completely dried in a Speedvac rotational vacuum concentrator instrument. The dried precipitate was dissolved in mQ water (100 μL). LCMS analysis showed the mass of the self-eluted model nucleic acid encoded library member (
Step 5.1 Oligonucleotide Attachment
5 nmol of 5′-azido modified single-stranded oligonucleotide was attached to the self-elution model compound synthesized in Example 3 (20 mg) following general procedure 5.
Step 5.1 MeDbz Linker Activation Step 1—Incubation with p-nitrophenyl Chloroformate
The functionalized TentaGel® beads (20 mg) with oligonucleotide attached were incubated with 100 mM p-nitrophenyl chloroformate in dichloromethane (600 μL) on a rotational shaker at room temperature for 30 min. The resin was washed with dichloromethane (3×600 μL).
Step 5.2 MeDbz Linker Activation Step 2—Incubation with N,N-diisopropylethylamine (DIPEA)
The functionalized beads were incubated with 8.7% (v/v) N,N-diisopropylethylamine (DIPEA) in dichloromethane (600 μL) on a rotational shaker at room temperature for 30-40 min. The resin was washed with dichloromethane (3×600 μL).
Step 5.3 Cleaving Group Deprotection
The functionalized beads were incubated in a solution of 100 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in 6% (v/v) triethylamine, 53% dimethylsulfoxide (DMSO) in mQ water with 0.01% (w/v) sodium dodecyl sulfate (SDS), pH 8-9, on a rotational shaker at 60° C. for 1 h. The beads were dried by centrifugation.
Step 5.4 Self-Elution of Model Nucleic Acid Encoded Library Member
The functionalized beads were incubated with a solution of 10% (v/v) N,N-diisopropylethylamine (DIPEA) in dichloromethane (150 μL) on a rotational shaker at 60° C. for 1 h. The beads were then dried by centrifugation. The beads were washed with a solution of 1 mM sodium carbonate (Na2CO3) in 49% DMSO in mQ water, with 0.01% SDS, pH 9, and the resulting solution collected by centrifugation was analyzed by LCMS (
Step 6.1 Dbz Linker Derivative Synthesis
4-Amino-3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]benzoic acid (Fmoc-Dbz-OH) (200 mg, 0.534 mmol, 1.0 equiv) was dissolved in 10 mL dimethylformamide (DMF) in a round bottom flask. Propargylamine (68.3 μL, 1.068 mmol, 2.0 equiv), N,N-diisopropylethylamine (DIPEA) (372 μL, 2.137 mmol, 4.0 equiv.), and N-[(Dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (407 mg, 1.068 mmol, 2.0 equiv.) were added and the solution was stirred at room temperature for 2 h. The reaction mixture was quenched with water. The aqueous component was extracted with ethyl acetate. The combined organic components were concentrated under reduced pressure. The crude product was purified by flash column chromatography (70% (v/v) ethyl acetate in hexane) to yield a white solid.
The above product was stirred in 20% (v/v) diethylamine in THF (10 mL) at room temperature for 5 h. The reaction mixture was concentrated under reduced pressure. The crude reaction product was used for attachment to solid support.
Preparation of a Nascent Library Member
Step 6.2 Coupling to N2-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N6-[(4-methylphenyl)diphenylmethyl]-L-lysine (Fmoc-Lys(Mtt)-OH)
Amine-functionalized solid support (functionalized ProMag® 1, Bangs Labarotories, Inc.) (50 μL) was coupled to N2-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N6-[(4-methylphenyl)diphenylmethyl]-L-lysine (Fmoc-Lys(Mtt)-OH) following general procedure 3.
Step 6.3 Fmoc Deprotection
The functionalized beads (50 μL) were Fmoc deprotected following general procedure 4.
Step 6.4 Coupling to 4-(Hydroxymethyl)benzoic acid (HMBA)
Amine-functionalized solid support (50 μL) was coupled to 4-(Hydroxymethyl)benzoic acid (HMBA) following general procedure 3.
Step 6.5 Coupling to (2S)-2-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-4-pentynoic acid (Fmoc-Pra-OH)
Alcohol-functionalized solid support (50 μL) was washed with dimethylformamide (200 μL). The functionalized beads were incubated with a solution of 100 mM diisopropylcarbodiimide (DIC), 5.76 mM DMAP and 80 mM (2S)-2-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-4-pentynoic acid (Fmoc-Pra-OH) in dimethylformamide (150 μL) on a rotational shaker at 4° C. for 4 h. The functionalized beads were washed with dimethylformamide (6×200 μL).
Step 6.6 Fmoc Deprotection
The functionalized beads (50 μL) were Fmoc deprotected following general procedure 4.
Step 6.7 Coupling to 1-(1,1-Dimethylethyl) Butanedioate Building Block
Amine-functionalized solid support (50 μL) was coupled to 1-(1,1-Dimethylethyl) butanedioate following general procedure 3.
Step 6.8 Mtt Deprotection and tBu Deprotection
The functionalized beads (50 μL) were incubated with 50% (v/v) trifluoroacetic acid in dichloromethane (600 μL) at room temperature on a rotational shaker for 3 min (step repeated 3×). The functionalized beads were washed with 10% (v/v) N,N-diisopropylethylamine (DIPEA) in dimethylformamide (3×200 μL), and then with dimethylformamide (3×200 μL).
Step 6.9 Oligonucleotide Attachment to Solid Support
5 nmol 5′-azido modified single-stranded oligonucleotide (synthesized in Example 1) was attached to the alkyne-functionalized beads (50 μL) following general procedure 6, using double the stated volumes.
Step 6.10 Small Molecule Alkyne Quenching
The functionalized beads (50 μL) after DNA attachment were subjected to CuAAC conditions with benzyl azide to quench any unreacted alkyne functional groups. The functionalized solid support (50 μL) was washed with dimethylsulfoxide (DMSO) (3×200 μL). The solid support was incubated in a mixture of 50 mM benzyl azide, 993 μM 1-(Phenylmethyl)-N,N-bis[[1-(phenylmethyl)-1H-1,2,3-triazol-4-yl]methyl]-1H-1,2,3-triazole-4-methanamine (TBTA), 945 μM copper sulfate (CuSO4), and 5.672 mM sodium ascorbate (NaAsc) 89% dimethylsulfoxide (DMSO) in mQ water (360 μL) on a rotational shaker at room temperature for 1 h. The solid support was washed with DMSO (3×200 μL). 5 μL of the functionalized solid support was kept for analysis.
Installation of Linker 2
Step 6.11 Coupling to 5-azidopentanoic acid (Reaction On-DNA on Solid Support)
50 mg/mL stocks of N3-(Ethylcarbonimidoyl)-N1,N1-dimethyl-1,3-propanediamine (EDC) and of 1-Hydroxy-2,5-dioxo-3-pyrrolidinesulfonic acid (S—NHS) were prepared in 100 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer pH 4.5. 40 μL of each stock was mixed with 120 μL of 450 mM 5-azidopentanoic acid in dimethylformamide (DMF) to result in a final volume of 200 μL. The solution was incubated for 15 min at room temperature. The solution was then added to the functionalized beads (45 μL) and the reaction was left on a rotational shaker at room temperature for 2 h. The functionalized beads were washed with dimethylformamide (DMF) (3×200 μL).
Step 6.12 Coupling to Reverse Dbz Linker 2 (Reaction On-DNA on Solid Support)
5 μL of the functionalized solid support was kept for analysis. The rest of the functionalized solid support (40 μL) was washed with dimethylformamide (DMF) (200 μL). The functionalized beads were incubated with a solution of 360 mM amine (reverse Dbz linker prepared in step 6.1), 360 mM HATU, and 1.50 M DIPEA in dimethylformamide (DMF) (100 μL) at 40° C. on a rotational shaker at room temperature for 1 h. The functionalized beads were washed with dimethylformamide (DMF) (3×200 μL).
Step 6.13 Oligonucleotide Cleavage of 10 μL Beads from Solid Support for LCMS Analysis
A portion of the functionalized solid support (10 μL) was incubated with 100 mM lithium hydroxide (LiOH) in 25% dimethylsulfoxide (DMSO) in mQ water (60 μL) for 1 h at 40° C. on a rotational shaker. The sample was filtered and analyzed by LCMS (
Step 6.14 Cyclisation by CuAAC
The remaining functionalized solid support (30 μL) was washed with dimethylsulfoxide (DMSO) (3×200 μL). The solid support was incubated in a mixture of 993 μM 1-(Phenylmethyl)-N,N-bis[[1-(phenylmethyl)-1H-1,2,3-triazol-4-yl]methyl]-1H-1,2,3-triazole-4-methanamine (TBTA), 945 μM copper sulfate (CuSO4), and 5.672 mM sodium ascorbate (NaAsc) in 89% DMSO in mQ water (270 μL) on a rotational shaker at room temperature for 1 h. The solid support was washed with DMSO (3×200 μL).
Self-Purification
Step 6.15 Cleavage of Linker 1
For the functionalized solid support (30 μL), the HMBA linker 1 was cleaved by 100 mM lithium hydroxide (LiOH) in 25% DMSO in mQ water (60 μL) at 40° C. for 1.5 h on a rotational shaker. LCMS analysis of this solution showed undesired products cleaved from solid support during this step (
Step 6.16 Cleavage of Linker 2 (Terminal Linker)
The Dbz linker 2 on the functionalized solid support (30 μL) was activated by incubation with 36 mM isopentyl nitrite in mQ water (200 μL) for 1.5 h at room temperature on a rotational shaker. The solid support was washed with mQ water (2×200 μL). The activated linker was cleaved in 100 mM lithium hydroxide (LiOH) in 25% DMSO in mQ water (60 μL). LCMS analysis showed the desired self-purified nucleic acid encoded library member (
Step 7.1 Coupling to N2-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N6-[(4-methylphenyl)diphenylmethyl]-L-lysine (Fmoc-Lys(Mtt)-OH)
Amine-functionalized solid support (functionalized ProMag® 1, Bangs Labarotories, Inc.) (50 μL) was coupled to N2-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N6-[(4-methylphenyl)diphenylmethyl]-L-lysine (Fmoc-Lys(Mtt)-OH) following general procedure 3.
Step 7.2 Fmoc Deprotection
The functionalized beads (50 μL) were Fmoc deprotected following general procedure 4.
Step 7.3 Coupling to 4-(Hydroxymethyl)benzoic Acid (HMBA)
Amine-functionalized solid support (50 μL) was coupled to 4-(Hydroxymethyl)benzoic acid (HMBA) following general procedure 3.
Step 7.4 Coupling to (2S)-2-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-4-pentynoic Acid (Fmoc-Pra-OH)
Alcohol-functionalized solid support (50 μL) was washed with dimethylformamide (DMF) (200 μL). The functionalized beads were incubated with a solution of 100 mM diisopropylcarbodiimide (DIC), 5.76 mM N,N-dimethyl-4-pyridinamine (DMAP) and 80 mM (2S)-2-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-4-pentynoic acid (Fmoc-Pra-OH) in dimethylformamide (150 μL) on a rotational shaker at 4° C. for 4 h. The functionalized beads were washed with dimethylformamide (DMF) (6×200 μL).
Step 7.5 Fmoc Deprotection
The functionalized beads (50 μL) were Fmoc deprotected following general procedure 4.
Step 7.6 Coupling to Fmoc-Dbz-OH
Amine-functionalized solid support (50 μL) was coupled to 4-Amino-3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]benzoic acid (Fmoc-Dbz-OH) following general procedure 3.
Step 7.7 Mtt Deprotection
The functionalized beads (50 μL) were incubated with 50% (v/v) trifluoroacetic acid in dichloromethane (600 μL) at room temperature on a rotational shaker for 3 min (step repeated 3×). The functionalized beads were washed with 10% (v/v) N,N-diisopropylethylamine (DIPEA) in dimethylformamide (3×200 μL), and then with dimethylformamide (3×200 μL).
Step 7.8 Oligonucleotide Attachment to Solid Support
5 nmol 5′-azido modified single-stranded oligonucleotide (synthesized in Example 1) was attached to the alkyne-functionalized beads (50 μL) following general procedure 6, using double the stated volumes.
Step 7.9 Small Molecule Alkyne Quenching
The functionalized beads (50 μL) after DNA attachment were subjected to CuAAC conditions with benzyl azide to quench any unreacted alkyne functional groups. The functionalized solid support (50 μL) was washed with DMSO (3×200 μL). The solid support was incubated in a mixture of 50 mM benzyl azide, 993 μM 1-(Phenylmethyl)-N,N-bis[[1-(phenylmethyl)-1H-1,2,3-triazol-4-yl]methyl]-1H-1,2,3-triazole-4-methanamine (TBTA), 945 μM copper sulfate (CuSO4), and 5.672 mM sodium ascorbate (NaAsc) 89% DMSO in mQ water (360 μL) on a rotational shaker at room temperature for 1 h. The solid support was washed with DMSO (3×200 μL). 5 μL of the functionalized solid support was kept for analysis.
Step 7.10 Coupling to 5-azidopentanoic Acid (Reaction On-DNA on Solid Support)
50 mg/mL stocks of N3-(Ethylcarbonimidoyl)-N1,N1-dimethyl-1,3-propanediamine (EDC) and of 1-Hydroxy-2,5-dioxo-3-pyrrolidinesulfonic acid (S—NHS) were prepared in 100 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer pH 4.5. 40 μL of each stock was mixed with 120 μL of 450 mM 5-azidopentanoic acid in dimethylformamide to result in a final volume of 200 μL. The solution was incubated for 15 min at room temperature. The solution was then added to the functionalized beads (45 μL) and the reaction was left on a rotational shaker at room temperature for 2 h. The functionalized beads were washed with dimethylformamide (3×200 μL). 5 μL of the functionalized solid support was kept for analysis.
Step 7.11 Fmoc Deprotection
The functionalized beads (40 μL) were Fmoc deprotected following general procedure 4, using 80% of the stated volumes.
Step 7.12 Coupling to 5-hexynoic Acid
The functionalized solid support (40 μL) was washed with DMSO (200 μL). The solid support was incubated in a solution of 5 mM 3-Hydroxy-3H-1,2,3-triazolo[4,5-b]pyridine (HOAt), 500 mM 5-hexynoic acid, and 50 mM N3-(Ethylcarbonimidoyl)-N1,N1-dimethyl-1,3-propanediamine (EDC) in DMSO (500 μL) at room temperature for 2 h on a rotational shaker. The solid support was washed with DMSO (3×200 μL). 10 μL of the functionalized solid support was kept for analysis.
Step 7.13 Cyclisation by CuAAC
Alkyne- and azide-functionalized solid support (30 μL) was washed with DMSO (3×200 μL).
The solid support was incubated in a mixture of 993 μM 1-(Phenylmethyl)-N,N-bis[[1-(phenylmethyl)-1H-1,2,3-triazol-4-yl]methyl]-1H-1,2,3-triazole-4-methanamine (TBTA), 945 μM copper sulfate (CuSO4), and 5.672 mM sodium ascorbate (NaAsc) 89% DMSO in mQ water (360 μL) on a rotational shaker at room temperature for 1 h. The solid support was washed with DMSO (3×200 μL).
Self-Purification
Step 7.14 Cleavage of Linker 1
For the functionalized solid support (30 μL), the HMBA linker 1 was cleaved by 100 mM lithium hydroxide (LiOH) in 25% DMSO in mQ water (60 μL) at 40° C. for 1.5 h on a rotational shaker.
Step 7.15 Cleavage of Linker 2 (Terminal Linker)
For the functionalized solid support (30 μL), the Dbz linker 2 was activated by incubation with 36 mM isopentyl nitrite in mQ water (200 μL) for 2 h at room temperature on a rotational shaker. The solid support was washed with mQ water (2×200 μL). The activated linker was cleaved in 100 mM lithium hydroxide (LiOH) in 25% DMSO in mQ water (60 μL). The cleavage solution was analyzed by LCMS (
Step 8.1 Coupling to Ethylenediamine
Carboxylic acid functionalized magnetic solid support (ProMag® 1, Bangs Labarotories, Inc.) (25 μL) was washed with DMSO (1×1 mL) and dimethylformamide (DMF) (2×1 mL). The functionalized beads were incubated with 360 mM HATU, 360 mM ethylenediamine and 1.1 M DIPEA in DMF (25 μL, 2×30 min). The functionalized beads were washed with dimethylformamide (3×200 μL).
Step 8.2 Coupling to 4-(Hydroxymethyl)benzoic Acid (HMBA)
Amine-functionalized solid support (25 μL) was coupled to 4-(Hydroxymethyl)benzoic acid (HMBA) following general procedure 3, using half of the respective stated volumes.
Step 8.3 Coupling to N2-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N6-[(4-methylphenyl)diphenylmethyl]-L-lysine (Fmoc-Lys(Mtt)-OH)
Amine-functionalized solid support (25 μL) was coupled to N2-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N6-[(4-methylphenyl)diphenylmethyl]-L-lysine (Fmoc-Lys(Mtt)-OH) following general procedure 3, using half of the respective stated volumes.
Step 8.4 Fmoc Deprotection
The functionalized beads (25 μL) were Fmoc deprotected following general procedure 4, using half of the respective stated volumes.
Step 8.5 Coupling to 1-(9H-Fluoren-9-ylmethyl) 5,8,11,14-tetraoxa-2-azaheptadecanedioate (Fmoc-PEG4-OH)
Amine-functionalized solid support (25 μL) was coupled to 1-(9H-Fluoren-9-ylmethyl) 5,8,11,14-tetraoxa-2-azaheptadecanedioate (Fmoc-PEG4-OH) following general procedure 3, using half of the respective stated volumes.
Step 8.6 Fmoc Deprotection
The functionalized beads (25 μL) were Fmoc deprotected following general procedure 4, using half of the respective stated volumes.
Step 8.7 Coupling to 5-hexynoic Acid
Amine-functionalized solid support (25 μL) was coupled to 5-hexynoic acid following general procedure 3, using half of the respective stated volumes.
Step 8.8 Mtt Deprotection
The functionalized beads (25 μL) were incubated with 50% (v/v) trifluoroacetic acid in dichloromethane (300 μL) at room temperature on a rotational shaker for 3 min (step repeated 3×). The functionalized beads were washed with 10% (v/v) N,N-diisopropylethylamine (DIPEA) in dimethylformamide (3×200 μL), and then with dimethylformamide (3×200 μL).
Step 8.9 Oligonucleotide Attachment to Solid Support
1 nmol 5′-azido modified single-stranded oligonucleotide (synthesized in Example 1) was attached to the functionalized solid support (25 μL) following general procedure 6, however without any lithium chloride.
Step 8.10 Ligation on Solid Support
The procedure for ligation was adapted from Pengpumkiat et al 2016. The functionalized solid support was washed with a solution of 10 mM Tris, 1 mM EDTA, 2 M NaCl, and 0.05% (v/v) Tween 20, pH 7.4 (1×200 μL). The functionalized solid support was then washed with mQ water (2×200 μL). To the functionalized solid support (25 μL) was added 1.9 nmol adaptor (Sequence 3) in 90 μL mQ water, and 10 μL of a solution of 100 mM Tris, 500 mM NaCl, 10 mM EDTA, pH 7.4. The mixture was heated to 95° C. for 10 min. The sample was left to cool to room temperature for 1 h. 1.5 nmol code (Sequence 4), 17 μL mQ water, 2 μL of 10× T4 ligase buffer (500 mM Tris-HCl, 100 mM MgCl2, 10 mM ATP, 100 mM DTT, pH 7.5, New England Biolabs) and 600 U (1 μL) T4 DNA ligase (New England Biolabs) were added to the mixture, and the ligation was performed at room temperature for 18 h. The functionalized solid support was washed with TE Buffer (10 mM Tris, 1 mM EDTA and 0.05% (v/v) Tween 20, pH 7.4).
Step 8.11 Oligonucleotide Cleavage from Solid Support for LCMS Analysis
The functionalized solid support (25 μL) was incubated with 100 mM lithium hydroxide (LiOH) in 25% DMSO in mQ water (60 μL) for 1 h at 40° C. on a rotational shaker. The sample was filtered and analysed by LCMS (
Carboxylic acid-functionalized solid support (functionalized ProMag® 1, Bangs Laboratories, Inc.) was used.
Step 9.1 Coupling to N-Boc-ethanolamine
Carboxylic acid functionalized magnetic solid support (50 μL) with was washed with DMSO (1×1 mL) and dimethylformamide (2×1 mL). The functionalized beads were incubated with 360 mM HATU, 360 mM N-Boc-ethanolamine and 1.1 M DIPEA in DMF (25 μL, 2×30 min). The functionalized beads were washed with dimethylformamide (DMF) (3×200 μL).
Step 9.2 Boc Deprotection
The functionalized beads (50 μL) were incubated with 50% (v/v) trifluoroacetic acid in dichloromethane (600 μL) at room temperature on a rotational shaker for 5 min. The beads were then again incubated with a fresh solution of 50% (v/v) trifluoroacetic acid in dichloromethane (600 μL) at room temperature on a rotational shaker for 15 min. The functionalized beads were washed with 1×PBS pH 7.4 (3×600 μL), and then with dimethylformamide (3×600 μL).
Step 9.3 Coupling to (2S)-2-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-4-pentynoic acid (Fmoc-Pra-OH)
Amine-functionalized solid support (50 μL) was coupled to (2S)-2-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-4-pentynoic acid (Fmoc-Pra-OH) following general procedure 3.
Step 9.4 Fmoc Deprotection
The functionalized beads (50 μL) were Fmoc deprotected following general procedure 4.
Step 9.5 Oligonucleotide Attachment to Solid Support
2 nmol 5′-azido modified single-stranded oligonucleotide (synthesized in Example 1) was attached to the alkyne-functionalized beads (50 μL) following general procedure 6, using double the respective stated volumes.
Step 9.6 Oligonucleotide Cleavage of 25 μL Beads from Solid Support for LCMS Analysis
Half of the functionalized solid support (25 μL) was incubated with 100 mM lithium hydroxide (LiOH) in 25% DMSO in mQ water (60 μL) for 1 h at 40° C. on a rotational shaker. The sample was filtered and analysed by LCMS.
Step 9.7 Coupling to 5-azidopentanoic Acid (Reaction On-DNA on Solid Support)
The remaining half of the functionalized solid support (25 μL) prepared in step 9.5 was used. 50 mg/mL stocks of N3-(ethylcarbonimidoyl)-N1,N1-dimethyl-1,3-propanediamine (EDC) and of 1-Hydroxy-2,5-dioxo-3-pyrrolidinesulfonic acid (S—NHS) were prepared in 100 mM 2-(N-morpholino) ethanesulfonic acid (MES) buffer pH 4.5. 20 μL of each stock was mixed with 60 μL of 450 mM 5-azidopentanoic acid in dimethylformamide (DMF) to result in a final volume of 100 μL. The solution was incubated for 15 min at room temperature. The solution was then added to the functionalized beads (25 μL) and the reaction was left on a rotational shaker at room temperature for 2 h. The functionalized beads were washed with dimethylformamide (DMF) (3×200 μL).
Step 9.8 Oligonucleotide Cleavage from Solid Support for LCMS Analysis
The functionalized solid support (25 μL) prepared in step 9.7 was incubated with 100 mM lithium hydroxide (LiOH) in 25% dimethylsulfoxide (DMSO) in mQ water (60 μL) for 1 h at 40° C. on a rotational shaker. The sample was filtered and analysed by LCMS.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20203475.7 | Oct 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/079294 | 10/21/2021 | WO |
